Introduction

Plumbagin is a naturally occuring compound originated from Plumbaginaceae, Droseraceae and Ebenceae families [13]. Crude extracts of plumbagin have been used to cure rheumatoid arthritis, dysmenorrhea, and toothache in the folk remedy [24]. Many reports have been suggested anti-bacterial, anti-fungal, anti-diabetic and anti-cancer effect of plumbagin [6, 7, 11, 19, 30]. Park et al. previously reported inhibitory function of plumbagin on Western type H. pylori strain (ATCC 43504), although the mechanism is yet to be elucidated [25].

Helicobacter pylori is a Gram-negative bacterium possessing its characteristic helical appearance. H. pylori primarily colonizes on human stomach and it has been reported to infect approximately half of the world population [33]. Infection of H. pylori on gastric mucosa is associated with various gastric disease including inflammation, chronic gastritis, peptic ulcer and gastric adenocarcinoma [4]. Moreover, because of its involvement in the gastric cancer development, H. pylori was classified as a group I carcinogen by WHO [10].

RNA polymerase is an indispensible protein responsible for transcription of DNA into RNA. Bacterial RNA polymerase consists of α, β and ω subunits (α2ββ’ω) [1]. β and β’ are the largest subunits of bacterial RNA polymerase possessing catalytic site, and β and β’ subunits in H. pylori exist as a single fused protein [1, 5]. α subunit binds to β and β’ subunits and stabilizes the complex, and ω subunit has both structural and functional role [1, 20]. In addition, there are σ factors which binds to core complex and facilitate promoter specific initiation of transcription [1, 28].

Various bacterial proteins are associated with virulence and pathogenesis of H. pylori such as toxins, ureases, adhesion proteins and flagella proteins. The most studied virulence factors of H. pylori are cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA). CagA protein is translocated to the host cells by Cag pathogenicity island (cagPAI) type IV secretion system [23]. Once injected, CagA proteins are phosphorylated by host cell Src kinases at its EPIYA motif and subsequently deregulate intracellular signaling transduction pathways, disrupt epithelial cell junctions, and induce inflammation [4, 9, 26, 29]. VacA has been known to induce cytoplasmic vacuole formation [3]. Moreover, VacA increases β-catenin level in the host cells by inhibiting glycogen synthase kinase 3-β and leads to uncontrolled cell growth [21].

Urease is one of the major virulence factors, which enables H. pylori to successfully colonize on gastric mucosa. The bacterium typically colonizes the strongly acidic mucosal lining of the stomach [16]. Therefore, increase in pH of the environment by secretion of urease allows the bacterium to persist in the hostile conditions [16]. The H. pylori urease comprises α and β that were known to form a dodecameric complex ((αβ)3)4 [16].

The adherence of H. pylori on the gastric mucosa is the first step of the bacterial colonization. Therefore, adhesins are also an important virulence factor that helps the bacteria to overcome the mucus and exfoliation of the epithelium [12, 27]. Adhesins of H. pylori belong to the largest outer membrane protein (OMP) family, namely, the Hop family which involves BabA, SabA, AlpA, AlpB, HopZ, and OipA [12].

In this study, we demonstrated minimal inhibitory concentration (MIC) of plumbagin on East Asian type reference strain of H. pylori (ATCC 49503) by agar dilution method and investigated expression of RNA polymerase subunits as well as various virulence factors after plumbagin treatment.

Materials and Methods

Materials

H. pylori reference strain was purchased from ATCC (ATCC49503, VA, USA). Mueller-Hinton broth, Mueller-Hinton agar and Brucella agar were purchased from Becton-Dickinson (MA, USA). Bovine serum was purchased from Gibco (NY, USA). Trizol reagent, random hexamer, and Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) were purchased from Invitrogen (CA, USA). Plumbagin and protease inhibitor cocktail were obtained from Sigma-Aldrich (MO, USA). Antibodies to detect CagA and VacA were purchased from Santa Cruz Biotechnology (TX, USA) and polyclonal antibody against whole H. pylori (ATCC 49503) was produced as previously described [14]. ECL kit was purchased from Thermo Scientific (MA, USA).

Bacterial culture

H. pylori were grown on the Brucella agar plate supplemented with 10% bovine serum at 37℃ for 72 h in a humidified atmosphere with 5% CO2. For cultivation in broth, bacterial colonies were collected and suspended in Mueller-Hinton broth supplemented with 10% bovine serum. The number of bacterial particles in the H. pylori suspension was set to MacFarland 0.33 and incubated at 37℃ for 72 h in a humidified atmosphere with 5% CO2.

Agar dilution method to determine MIC

H. pylori grown on the Brucella agar plate were collected and suspended in saline. The number of bacterial particles in the H. pylori suspension was set to MacFarland 4.0. Thirty μl of the bacterial suspension was placed on the Mueller-Hinton agar supplemented with 10% bovine serum including indicated concentrations of plumbagin. The bacteria were incubated for 72 h and MIC was determined based on the lowest concentration at which inhibition of growth was observed.

Broth dilution method to determine MIC

H. pylori grown on the Brucella agar plate were collected and suspended in Mueller-Hinton broth supplemented with 10% bovine serum. The number of bacterial particles in the H. pylori suspension was set to MacFarland scale 0.5. Various concentrations of plumbagin (125 nM−4 μM) were treated and the bacteria were incubated for 72 h and final optical density (600 nm wave length) of the bacterial suspension was measured by spectrophotometry.

RT-PCR (reverse transcription-polymerase chain reaction)

H. pylori was grown in the Mueller-Hinton broth for 72 h. Cultured H. pylori were washed twice with PBS and total RNA was extracted using Trizol reagent as described in the manufacturer’s instructions. cDNA was synthesized by reverse transcription with 2 μg total RNA, 0.25 μg of random hexamer and 200 U of MMLVRT for 50 min at 37℃ and 15 min at 70℃. Subsequent PCR amplification using 0.2 U of Taq polymerase was performed in a thermocycler using specific primers. The PCR primer sequences used in this study are listed in Table 1.

Table 1.List of primer sequences used for RT-PCR.

Westernblotting

Cultured H. pylori were washed twice with PBS and then lysed with RIPA buffer containing protease inhibitor cocktail. The cell lysates were sonicated for 1 min and incubated on ice for 10 min. The cell lysates were then centrifuged and the supernatants were collected. The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated with optimal concentrations of primary antibody at 4℃ overnight and then incubated with the appropriate secondary antibody (anti-mouse or anti-rabbit) for 1 h at room temperature. The immunolabeled proteins were visualized using ECL. Anti-H. pylori antibody was used as an internal control.

Statistical analysis

Data in the bar graphs are presented as mean ± standard error of mean (SEM). All the statistical analyses were performed using GraphPad Prism 5.02 software (GraphPad Software, CA, USA). All the data were analyzed by unpaired Student’s t-test and p < 0.05 was considered to be statistically significant (*p < 0.05, **p < 0.01 and ***p < 0.001).

Results

Inhibitory effect and MIC of plumbagin against H. pylori

Park et al. previously reported the inhibitory effect of plumbagin on H. pylori growth. However, the inhibitory mechanism and effect of plumbagin on virulence factors of H. pylori have not been studied yet. In this study, therefore, we investigated whether plumbagin treatment has an influence on the expression of virulence factors in H. pylori. First, we investigated inhibitory effect and MIC of plumbagin on H. pylori ATCC 49503 strain. To define MIC, agar dilution method was performed. Various concentrations of plumbagin (250 nM−64 μM) was diluted in Mueller-Hinton agar and 30 μl of bacterial suspension set to McFarland scale 4.0 was placed on the agar, then MIC was defined after 72 h of incubation. In the agar dilution test result, MIC of plumbagin on H. pylori ATCC 49503 strain was 8 μM which was lower than 4 μg/ml (approximately 21.3 μM) as previously reported by Park et al. (Fig. 1). The discordance of MIC was presumably came from the difference of bacterial strain. Furthermore, we also determined MIC of plumbagin by broth dilution method and found that MIC in the broth dilution test was 1 μM which was lower than the result from agar dilution test. In the agar dilution method, drug should diffuse to the bacteria growing on the surface of the medium whereas drug in the broth can directly react with bacteria. This is presumably the reason why MIC in the broth dilution method showed higher than that of agar dilution method. Based on this result, we reconfirmed the inhibitory effect of plumbagin on H. pylori and especially we defined MIC of plumbagin on East Asian type strain of H. pylori (ATCC 49503).

Fig. 1.Demonstration of minimal inhibitory concentration of plumbagin against H. pylori by agar dilution method. (A) Thirty μl of bacterial suspension set to McFarland scale 4.0 (1.2 × 109/ml) was placed on the Mueller-Hinton agar supplemented with 10% bovine serum and treated with indicated concentrations of plumbagin. MIC of plumbagin against H. pylori was determined after 72 h of incubation, (B) H. pylori suspension was set to MacFarland scale 0.5 in Mueller-Hinton broth supplemented with 10% bovine serum. Indicated concentrations of plumbagin (125 nM−4 μM) were treated and the bacteria were incubated for 72 h. Final optical density (600 nm wave length) of the bacterial suspension was measured by spectrophotometry and determined MIC.

Effect of plumbagin on RNA polymerase of H. pylori

RNA polymerase is indispensible enzyme for living organisms necessary for transcription and subsequent synthesis of proteins. RNA polymerase in H. pylori consists of several subunits including α subunit, β subunit, σ-70 factor, and σ-64 factor that are respectively encoded by rpoA, rpoB, rpoD, and rpoN. Therefore, we investigated expression of the RNA polymerase subunits in H. pylori after plumbagin treatment. Protein and RNA were hardly extractable after 3 days of plumbagin treatment because plumbagin inhibits bacterial growth in 3 days, thus we conducted experiments in a time dependent manner in a shorter time period. H. pylori was inoculated in Mueller-Hinton broth supplemented with 10% bovine serum and treated with plumbagin for various time periods (0, 1, 3, 6, and 12 h) then RNA was extracted and subjected to RT-PCR. The final concentration of plumbagin was set to 2 μM, because 2 μM was enough to completely inhibit the bacterial growth in the broth. In the results, we found that expression of rpoA was decreased by plumbagin treatment in H. pylori (Fig. 2). The expression of rpoA began to decrease in 12 h of plumbagin treatment (Fig. 2). In E.coli, the four RNA polymerase subunits assemble in a sequential manner (α – α2 – α2β – α2ββ') [15]. RNA polymerase α subunit takes part in the initiation of the enzyme assembly, because dimerization of α subunits is the first step in assembly of the RNA polymerase suggesting the key role of α subunit in assembly of the enzyme [15]. Therefore, decreased expression of RNA polymerase α subunit seems to be sufficient to inhibit the function of RNA polymerase and subsequent growth of H. pylori, although expressions of other RNA polymerase subunits were not changed by plumbagin treatment. This result indicates that inhibitory mechanism of plumbagin on H. pylori growth is at least in part associated with reduced expression of RNA polymerase α subunit.

Fig. 2.Effect of plumbagin on the expression of RNA polymerase subunits in H. pylori. (A) H. pylori suspension set to McFarland scale 0.33 (1 × 108/ml) was treated with 2 μM of plumbagin for indicated time periods. RNA was harvested and subjected to RT-PCR to investigate the expression of RNA polymerase subunits (rpoA, rpoB, rpoD, and rpoN). galE were used as an internal control, (B) Density of the bands were illustrated as a graph, and the results from triplicate experiments were analyzed by unpaired Student’s t-test (*p < 0.05, **p < 0.01 and ***p < 0.001).

Effect of plumbagin on virulence factors of H. pylori

There are various virulence factors necessary for H. pylori to colonize on gastric mucosa, to survive in the hostile environment, or to induce pathological changes in the host. The best investigated virulence factors in H. pylori are CagA and VacA toxins, and both toxins are tightly associated with bacterial pathogenesis. Therefore, we investigated whether plumbagin affects expression of the toxins in H. pylori. The expression levels of cagA and vacA genes were evaluated by RT-PCR after treatment of plumbagin in H. pylori. Moreover, expression of secA, which is responsible for secretion of VacA, was also investigated after plumbagin treatment. In the results, we found that mRNA expression of both cagA and vacA were decreased by plumbagin in H. pylori, but secA expression was not changed (Fig. 3A). In the Westernblot, the protein levels of CagA and VacA toxins were also decreased in 18 h following decrease of their mRNA expression (Fig. 3B). These results suggest that plumbagin inhibits synthesis of CagA and VacA toxins in H. pylori, although it does not affect secretion of VacA mediated by SecA.

Fig. 3.Effect of plumbagin on the expression of H. pylori virulence factors. H. pylori suspension set to McFarland scale 0.33 (1 × 108/ml) was treated with 2 μM of plumbagin for indicated time periods. (A) mRNA expression level of cagA and vacA toxins and secA after plumbagin treatment, (B) Protein level of CagA and VacA toxins after plumbagin treatment. Polyclonal anti-H. pylori antibody was used as an internal control (Control), (C) mRNA expression level of ureases after plumbagin treatment, (D) mRNA expression level of adhesins after plumbagin treatment. Density of the bands were illustrated as a graph, and the results from triplicate experiments were analyzed by unpaired Student’s t-test (*p < 0.05, **p < 0.01 and ***p < 0.001).

H. pylori possess virulence factors for successful colonization and survival in the host gastric mucosa. Ureases are one of the essential virulence factors for H. pylori to infect in the host gastric mucosa. This is because H. pylori utilize ureases as a strategy to survive from the acidic condition in the stomach. Therefore, inhibition of urease synthesis will seriously threat the survival of H. pylori from the hostile environment. Our investigations on expression of the urease subunits showed that ureA mRNA level is decreased by plumbagin, but ureB expression remained constant (Fig. 3C). Furthermore, we investigated expression of adhesins, which are also closely associated with colonization of the bacteria, in H. pylori after plumbagin treatment. Among the adhesins examined in this study, alpA mRNA level was reduced by plumbagin treatment (Fig. 3D). Collectively, our data regarding expression of various virulence factors of H. pylori suggest that plumbagin treatment on H. pylori inhibits expression of cagA, vacA, ureA and alpA all of which are necessary for bacteria to successfully infect with and to subsequently induce disease in the host.

Discussion

In this study, we confirmed inhibitory effect of plumbagin on East Asian type H. pylori strain (ATCC49503) and defined MIC by agar dilution method. Next, we investigated expression of RNA polymerase subunits in H. pylori and found that plumbagin inhibits expression of RNA polymerase α subunit. Furthermore, we also investigated expression of various virulence factors in H. pylori and found that expressions of cagA, vacA, ureA and alpA were decreased by plumbagin treatment in H. pylori.

Although anti-microbial activity of plumbagin has been studied in a few types of H. pylori strains (ATCC 43504, BCRC 17021, BCRC 17023, BCRC 17026, BCRC 17027 and BCRC 15415), the mechanism in which plumbagin inhibits growth of H. pylori has not been reported yet [25, 32]. To elucidate the inhibitory mechanism, we investigated expression of the molecules associated with DNA replication or transcription in H. pylori after plumbagin treatment. DNA replication is a crucial step for survival and propagation of living organisms. At first, therefore, we investigated whether plumbagin has an influence on the expression of the molecules involved in the H. pylori replication. In bacteria, DNA replication comprises three steps: initiation, elongation and termination [31]. Various molecules take part in the replication process that include a chromosomal replication initiator protein (DnaA), DNA helicase (DnaB), DNA polymerase III core polymerases (DnaE, DnaQ and HolE), sliding clamp (DnaN) and multiprotein clamploaders (DnaX, HolA, HolB, HolC and HolD) all of which are necessary to appropriately function as single multicomponent machinery [22]. We treated plumbagin on H. pylori and investigated expression of dnaA, dnaB, dnaE, dnaN, dnaQ, and holB by RT-PCR, but no change was observed among the molecules we investigated (Supplementary Fig. 1). Transcription is also tightly associated with growth of living organisms because it is indispensible for synthesis of protein in the end. In the investigation on the RNA polymerase subunits, which are key molecules for transcription, we found that mRNA level of α subunit (rpoA) in H. pylori was reduced by plumbagin treatment (Fig. 2). α subunit, which is one of the core subunits of RNA polymerase, binds to β subunit to initiate assembly of functional RNA polymerase and stabilizes the structure of the RNA polymerase [15]. Based on our data, therefore, decrease of rpoA expression is at least in part involved in the inhibition of H. pylori growth by plumbagin.

As many virulence factors in H. pylori are associated with successful infection of the bacteria or bacteria-induced pathogenesis, down-regulation of virulence factors may decrease chance for H. pylori to colonize on the host gastric epithelium as well as reducing pathogenesis by the bacteria. Among the various virulence factors, we found plumbagin treatment inhibited expression of cagA, vacA, ureA, and alpA in H. pylori. CagA and VacA toxins are closely associated with tumorigenesis by H. pylori. They disrupt intracellular signaling in host cells that lead to uncontrolled growth of the cells and inflammatory responses [9, 21]. These reports imply that decreased expression of CagA and VacA toxins by plumbagin treatment will alleviate the pathogenesis in gastric mucosa by H. pylori. Urease is an essential virulence factor of H. pylori because it is closely associated with survival of the bacteria in the acidic environment of gastric mucosa [16]. In this study we investigated expression of two urease subunits, ureA and ureB, and found that plumbagin treatment reduced ureA expression. The two urease subunits form dodecameric complex ((αβ)3)4 to function as an enzyme [16]. Therefore, reduced expression of one of them may decrease the enzyme activity which in turn impedes survival of H. pylori in the hostile condition of host stomach. There are adherence associated proteins enabling H. pylori to adhere to mucosal epithelial cells during the first step of bacterial colonization. AlpA is an adhesin involved in adhering to gastric tissue, and we found plumbagin treatment down-regulated alpA expression in H. pylori [12]. Therefore, decreased expression of AlpA by plumbagin treatment may reduce the colonization of H. pylori. We further investigated expression of flagella molecules but they were not affected by plumbagin treatment (Supplementary Fig. 1). Collectively, plumbagin treatment reduced expression of various virulence factors in H. pylori and these results imply that plumbagin may inhibit the colonization and pathogenesis by H. pylori.

The first-line regimen currently recommended for eradication of H. pylori is triple therapy including clarithromycin, amoxicillin and proton pump inhibitor [18]. However, numerous reports are alarming the limitation of current empirical regimen for eradication of H. pylori, because of the prevalence of clarithromycin resistance worldwide and decreased eradication rate of H. pylori by first-line therapy [8, 34, 35]. In particular, clarithromycin resistance in Asia has been surprisingly increased from 15.28% in 2009 to 32.46% in 2014 according to the recent review by Ghotaslou et al. [8]. In addition, resistance rate against levofloxacin has been increased and metronidazole resistance rate is also high in H. pylori [8]. Continuous increase of the antimicrobial resistance of H. pylori corresponding to the use of the antibiotics is a significant limitation for effective eradication of H. pylori in the future [17]. These reports collectively show the importance of surveillance on antibiotic resistance and selection of appropriate antibiotic regimen as well as development of a new therapeutic agent for eradication of H. pylori. Therefore, natural compounds such as plumbagin can be potentially suggested as an alternative choice for eradication of H. pylori in the future, although further studies seem to be necessary to completely understand the inhibitory mechanism of plumbagin on H. pylori and to confirm the effectiveness in vivo.