Helicobacter pylori is a gram-negative spiral-shaped bacterium which possess its characteristic helical appearance. H. pylori has been known to primarily colonize on human gastric epithelium. Approximately half of the world population harbor H. pylori and it has been suggested that infection of H. pylori generally occurs in childhood and transmitted from family members (Tharmalingam et al., 2016). Numerous reports suggested that various gastric diseases such as gastric inflammation, chronic gastritis, peptic ulcer and gastric adenocarcinoma are associated with H. pylori infection (Cover and Blaser, 2009). Epidemiologically, over 3 billion people are infected by this bacterium and develop persistent stomach inflammation, which lasts for decades unless treated with antibiotics. Moreover, World Health Organization classified H. pylori as a class I carcinogen due to the organism causes gastric cancer (Peek and Blaser, 2002).

Riboflavin (vitamin B2) is an essential water-soluble substance present in a wide variety of foods and required for normal cellular functions, including growth and development in all aerobic forms of life. Riboflavin is known as cofactors for enzymes involved in oxidation-reduction reactions, and has a role in the oxidative folding of proteins in the endoplasmic reticulum (Hoppel and Tandler, 1975; Tu et al., 2000).

Its most important biological forms, flavin adenine dinucleotide (FAD) and flavin mononucleotide, participate in a range of redox reactions, some of which are essential to the function of aerobic cells (Powers, 2003). Previous studies have shown that riboflavin can protect microbial keratitis. Riboflavin/UVA was effective against Staphylococcus aureus, Staphylococcus epidermidis, drug-resistant Streptococcus pneumoniae, methicillin-resistant S. aureus, multidrug-resistant Pseudomonas aeruginosa, and drug-resistant Streptococcus pneumoniae and had the potential for antimicrobial properties in vitro (Martins et al., 2008). Moreover, riboflavin has been found to increase the survival rate of mice suffering endotoxin-induced sepsis and Gram-negative and Gram-positive bacterial infection (Shih et al., 2010). In addition, riboflavin was also involved in the enhancement of antitumor activity of many anticancer drugs, as well as in the activation of the immune system to kill tumor cells (Zhu et al., 2006; de Souza Queiroz et al., 2007; Santos et al., 2007).

Based on the previous reports suggesting anti-microbial activity of riboflavin, we hypothesized that riboflavin has a potential role in the growth inhibition of H. pylori. To prove this hypothesis, we investigated the effect of riboflavin against H. pylori growth. We determined minimal inhibitory concentration (MIC) of riboflavin against H. pylori using agar dilution method and broth dilution method. Then expressions of DNA replication genes in H. pylori were investigated after riboflavin treatment. We also evaluated cytotoxicity of riboflavin on mammalian cells.

Bacterial and mammalian cell culture

The H. pylori reference strain ATCC 49503 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). H. pylori were grown on brucella agar plates (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 10% bovine serum (BRL Life Technologies, Grand Island, NY, USA) under microaerophilic and 100 percent humidity conditions at 37℃ and inspected after three days.

AGS gastric adenocarcinoma cells (ATCC CRL-1739) were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in Dulbeccós modified Eaglés medium (DMEM, BRL Life Technologies) supplemented with 10% fetal bovine serum (FBS, BRL Life Technologies) and streptomycin-penicillin (100 μg/mL and 100 IU/mL) (BRL Life Technologies). Cells were incubated at 37℃ in a humidified atmosphere with 5% CO₂.

Agar dilution method to determine MIC

H. pylori colonies grown on brucella agar plates were collected and suspended in saline. The number of bacterial particles in the H. pylori suspension was set to McFarland 2.0 (6×10⁸/mL), 10 μL of the bacterial suspension was placed on the Mueller-Hinton agar (BD Biosciences) sup ple mented with 10% bovine serum including indicated concentrations of riboflavin. All solutions were prepared in such a manner that the final dimethylsulfoxide (DMSO) concentration was the same in all treatments. The bacteria were incubated for 72 hours and the MIC was determined based on the lowest concentration at which inhibition of growth was observed in a previous report (Yang et al., 2019).

Broth dilution method to determine MIC

H. pylori suspension set to McFarland 0.33 was prepared in Mueller-Hinton broth (BD Biosciences) supplemented with 10% bovine serum. Various concentrations of riboflavin were added and the bacteria were grown at 37℃ for 72 h in a humidified atmosphere with 10% CO₂. All solutions were prepared in such a manner that the final DMSO concentration was the same in all treatments. The final optical density (at 600 nm wavelength) of the bacterial suspension was measured by spectrophotometry in a previous report (Woo et al., 2020).

RT-PCR (reverse transcription-polymerase chain reaction)

Cultured H. pylori were collected and washed twice with PBS. After washing, total RNA was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as described in the manufacturer's instructions. cDNA was synthesized by reverse transcription with 2 μg of total RNA, 0.25 μg of random hexamer (Invitrogen) and 200 U of Moloney murine leukemia virus reverse transcriptase (MMLV-RT, Invitrogen) for 50 minutes at 37℃ and 15 minutes at 70℃. Subsequent PCR amplification using 0.2 U of Taq polymerase (SolGent^TM, Daejeon, Korea) was performed in a thermocycler using specific primers. The PCR primer sequences used in this study and PCR conditions are described in Table 1. Amplified PCR products were analyzed using 2% agarose gel electrophoresis with Tris-acetate-EDTA (TAE) buffer, stained by ethidium bromide (EtBr) for 10 minutes and destained in distilled water for 20 minutes. Gel images were taken using Molecular Image Gel Doc^TM XR+ system (Bio-Rad, Hercules, CA, USA). The band intensity of PCR product was analyzed with the ImageLab software (Bio-Rad).

Table 1

List of primer sequences and PCR conditions for RT-PCR

Primers	Sequences (5'-3')		Product length (bp)	Anncaling temperature (℃)	Cycles	Reference*
	Forward	Reverse
DnaA	GGGCATGACTTAGCGGTTA	TTAACGAATTGCACGCCAAC	128	55	27	*
DnaB	AATGGGCCGTTTATCGTCTC	CAAATCCGCTTGCAACTACG	231	55	27
DnaE	AATCCACCGGCTCCAAATAC	GCCAAACAAGTGTGGGAGTA	184	55	27
DnaN	GTTAGCGGTGGTTGAAAACG	CGGTTTCGCTATGCTCAGAA	233	55	27
DnaQ	CGCATGAAGCTTTGCAAGAA	GCATAGGCTCTATGGCTGAC	244	55	27
PloA	TCATCATCATTGCCGACTGG	GTCATGCGCAAACACATTCA	134	55	24
HolB	GCCCTTGAAATCGTGCTTAC	ATGATGAGAGCTACCCGACA	196	55	25
RpoB	TTTAGGTAAGCGCGTGGATT	AATCAGCTTTGGATGGAACG	301	59	24
POLA1	GCCAGTTTTGGGCTGGTTG	GTTCGGTTTTGTCACTGCGA	458	57	29
MCM2	GGCGGAATCATCGGAATCCT	ATCATCCAGAGCCAGTCCCT	295	57	29
GAPDH	CGGGAAGCTTGTCATCAATGG	GGCAGTGATGGCATGGACTG	349	55	24	(Lee et al., 2015a)

*The primers without reference are designed in this study

Cell viability assay

AGS cells (1.5×10⁴/well) were plated in 96-well plates. After 24 h, cells were treated with various concentrations of riboflavin dissolved in DMSO. All solutions were prepared in such a manner that the final DMSO concentration was the same in all treatments. The cells were then further incubated for 24 h before cell viability was measured by WST (water soluble tetrazolium salts) assay using EZ-Cytox cell viability assay kit as described in a previous report (Yeon et al., 2019). Briefly, 10 μL of WST solution was added to the cultured media and incubated in the CO₂ incubator for 2 h. Absorbance at 450 nm was measured by spectrophotometer.

Statistical analysis

Data in the graphs have been presented as mean ± standard error of mean (SEM). Statistical analyses were performed using GraphPad Prism 5.02 software (GraphPad Software, San Diego, CA, USA). Data were analyzed by unpaired Student's t-test and P < 0.05 was considered be statistically significant (*P < 0.05, **P < 0.01 and ***P < 0.001).

MIC of riboflavin against H. pylori

To demonstrate inhibitory effect and MIC of riboflavin against H. pylori, we performed agar dilution test. MH agars containing 0.125~2 mM of riboflavin were prepared and 30 μL of H. pylori suspension set to McFarland 2.0 was placed on the MH agar. After then, H. pylori was incubated at 37℃ and 10% CO₂ condition for 72 h. In the result, we found that H. pylori growth was inhibited by 1 mM or higher concentration of riboflavin in the agar dilution test (Fig. 1A). In addition, we further conducted broth dilution test to define the MIC of riboflavin against H. pylori in broth condition. H. pylori set to McFarland 0.33 was grown in the MH broth containing 0.125~2 mM of riboflavin for 72 h. In the broth dilution test, MIC of riboflavin against H. pylori was shown to be 1 mM (Fig. 1B). Based on these results, we demonstrated that riboflavin suppresses the growth of H. pylori and MIC was 1 mM in agar dilution test and broth dilution test.

Fig. 1. Determination of the MIC of riboflavin against an H. pylori ATCC 49503 strain by broth dilution. (A) H. pylori suspended in saline was set to McFarland 2.0 (6×10⁸/mL) and plated on Muller-Hinton agar including indicated concentrations of riboflavin. After 72 h of incubation, MIC was determined. (B) H. pylori was set to McFarland 0.33 (1×10⁸/mL) and incubated in Muller-Hinton broth including indicated concentrations of riboflavin for 72 h. And then, absorbance of medium was detected at 600 nm wavelength to determine the MIC. Data were from triplicate experiments and analyzed by Student's t-test (***P < 0.001).

Down-regulation of replication-associated molecules in H. pylori by riboflavin

Various factors are involved in the growth of bacteria and, especially the expression of replication-associated molecules is one of the most important factors to the bacterial growth. To explain the mechanism by which riboflavin suppresses growth of H. pylori, we investigated whether riboflavin has an influence on the replication-associated molecules of the bacteria. On the basis of the MIC acquired from broth dilution test, H. pylori was treated with 0.25~0.5 mM of riboflavin. RNA was isolated from the samples and analyzed by RT-PCR to detect mRNA expression level of replication machineries. In the result, we found that treatment with riboflavin led to down-regulation of mRNA expression levels of polA, dnaB in a riboflavin dose dependent manner, though expression of the other genes remained constant (Fig. 2A). Reduction of polA expression induced by riboflavin was statistically significant in the dose of 0.5 mM (P = 0.0080) or higher concentration (Fig. 2B) and dnaB expression was statistically significant in the dose of 0.25 mM (P = 0.0201) or higher concentration (Fig. 2B). dnaB is a bacterial helicase and polA is a DNA polymerase I both of which paly the crucial role in a process of bacterial replication.

Fig. 2. Downregulation of replication machineries expressions in H. pylori treated with riboflavin. (A) H. pylori was treated with indicated concentrations of riboflavin for 72 h and RNA was harvested. Isolated RNA samples were analyzed by RT-PCR to detect mRNA expression level of replication machineries (polA, dnaB, dnaA, dnaE, dnaN, dnaQ and holB). (B) Density of polA and dnaB bands were measured and illustrated as graphs. Data were from triplicate experiments and analyzed by Student's t-test (*P < 0.05, **P < 0.01 and ***P < 0.001).

Therefore, these findings demonstrate that riboflavin may inhibit H. pylori growth by down-modulating polA and dnaB expression.

Evaluation of cytotoxicity of riboflavin on human gastric epithelial cells

As abovementioned, we confirmed that riboflavin suppresses the growth of H. pylori. Subsequently, we investigated whether riboflavin has cytotoxicity to human cells. We used AGS, a gastric adenocarcinoma cell line, in this experiment, because H. pylori colonizes on the human gastric epithelium. AGS cells treated with 0.25~2 mM of riboflavin were incubated for 24 h. After incubation, we measured cell viability of AGS cells by WST assay. As a result, AGS cells treated with riboflavin did not show statistically significant decrease of cell viability within the range of concentration used in this experiment (Fig. 3A). Additionally, we conducted RT-PCR to elucidate whether riboflavin has an influence on the replication machinery of AGS cells. The target genes were POLA1 and MCM2 in eukaryotic cells which are corresponding to the prokaryotic replication machinery, polA and dnaB. As a result, mRNA expressions of POLA1 and MCM2 were not decreased by 0.25~1 mM of riboflavin treatment in AGS cells (Fig. 3B). Thus, this result indicates that riboflavin can suppress the replication machinery of H. pylori but not on the corresponding replication machinery in AGS cells. Taken together, these findings demonstrate that riboflavin does not show cytotoxicity on AGS cells in the range of dose completely inhibited growth of H. pylori.

Fig. 3. Cytotoxicity evaluation of riboflavin on AGS cells. (A) AGS cells were placed on 96 well plates and treated with indicated concentrations of riboflavin. After 72 h, cell viability was measured by WST assay. (B) AGS cells were treated with indicated concentrations of riboflavin and RNA was harvested. Isolated RNA samples were analyzed by RT-PCR to detect mRNA expression level of eukaryotic replication machineries (MCM2 and POLA1).

In this study, we demonstrated inhibitory function and mechanism of riboflavin against H. pylori. MIC of riboflavin against H. pylori was 1 mM in the agar dilution test and broth dilution test, respectively. To elucidate the inhibitory mechanism of riboflavin, we investigated expressions of replication-associated molecules of the bacteria and found that polA and dnaB mRNA levels were down-regulated by riboflavin. Furthermore, riboflavin did not inhibit cell viability and mRNA expressions of POLA1 and MCM2 in AGS cells within the range of concentration completely inhibited growth of H. pylori.

DNA replication is indispensable for survival and proliferation of living organisms including bacteria. In prokaryotes, DnaA is a highly conserved protein in all bacteria which activates initiation of DNA replication. It binds to OriC site which has AT rich region within the replication origin. DnaA provides docking platform for DnaB, the replicative DNA helicase, in a complex with DnaC (Chodavarapu and Kaguni, 2016). H. pylori does not have DnaC but DnaB binds to DNA double strand and function as an helicase without help of DnaC (Soni et al., 2005). DnaB helicases load on to the unwound region and migrates along the single-stranded DNA, unwinding the DNA as it travels in the 5'-3' direction (Nitharwal et al., 2011). The interaction of DnaB with primase (DnaG) leads to the primer formation (Chodavarapu and Kaguni, 2016). The elongation phase begins when DNA polymerase III loads on to the 3' termini of the primer synthesized by DnaG (Nitharwal et al., 2011). DNA polymerase III is composed of core polymerases (DnaE, DnaQ and HolE), sliding clamp (DnaN) and multiprotein clamp-loader (DnaX, HolA, HolB, HolC and HolD) all of which are essential for appropriate function as a single machinery (Nitharwal et al., 2011). DNA polymerase I is encoded by polA gene. The major role of DNA polymerase I is to repair any damage with DNA, and to connect Okazaki fragments by deleting RNA primers and replacing the strand with DNA (Joyce et al., 1982). In our experiment, we found that riboflavin down-regulated mRNA expression of dnaB and polA genes. Both DNA helicase and DNA polymerase I are indispensable for replication and survival of the bacteria. Thus, the down-regulation of dnaB and polA explains how riboflavin inhibited growth of H. pylori.

Currently, triple therapy using clarithromycin, amoxicillin and proton pump inhibitor are recommended as the first-line regimen for eradication of H. pylori (Lee et al., 2015b). However, numerous reports warning the prevalence of clarithromycin resistance and decrease of eradication rate by first-line therapy worldwide (Lee et al., 2013; Ghotaslou et al., 2015; Wu et al., 2015; Zhang et al., 2015). Clarithromycin resistance rate in Asia has been markedly increased from 15.28% in 2009 to 32.46% in 2014 (Ghotaslou et al., 2015). Moreover, resistance rate of other antibiotics such as levofloxacin and metronidazole have also been increased in H. pylori (Ghotaslou et al., 2015). Increase of the antibiotic resistance seems unavoidable as long as we use the antibiotics for eradication of H. pylori, thus there should be continuous efforts for surveillance on antibiotic resistance rate and appropriate selection of antibiotic regimens. Furthermore, development of new therapeutic agents to help eradication of H. pylori is also necessary to overcome the prevalence of antibiotic resistance at this moment and in the future. There have been reports that riboflavin, a natural compound, inhibits the growth of various Gram-positive and negative bacteria, but no studies have been reported on its effect on the growth and inhibitory mechanisms of H. pylori. Therefore, this study confirmed the results that riboflavin inhibited growth by downregulation of the replication factors of H. pylori. However, as report on the correlation between riboflavin receptors in patients infected with H. pylori have been published, various possibilities for inhibitory mechanisms cannot be excluded (Matnuri et al., 2015). Therefore, further studies seem to be necessary to understand the inhibitory function and mechanism of riboflavin in H. pylori in detail, and its toxicity and effectiveness need to be confirmed in vivo.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Sciences and ICT) (No. 2019R1G1A1100451).

The authors declare that there is no conflict of interests regarding the publication of this article.