Possible mechanisms of the antimicrobial effects of polypeptide‑enriched Gastrodia elata Blume extracts

  • Authors:
    • Fange Kong
    • Xueying Cai
    • Siyu Zhai
    • Ruochen Wang
    • Xiaoyi Zheng
    • Yue Ma
    • Hui Bi
    • Di Wang
  • View Affiliations

  • Published online on: September 25, 2019     https://doi.org/10.3892/mmr.2019.10706
  • Pages: 4723-4730
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Abstract

The present study aimed to evaluate the antimicrobial activity and the possible mechanisms of activity of polypeptide‑enriched Gastrodia elata extracts (GEP) against the gram‑negative bacteria Escherichia coli and Pseudomonas aeruginosa, the gram‑positive bacterium Staphylococcus aureus and the fungus Candida albicans. The antimicrobial activity of GEP was first confirmed by determining the minimum inhibitory concentration by growth curve analysis. GEP was found to damage the cell wall and membrane of the microorganisms tested, as revealed by the morphological changes visible through scanning electron microscopy, and by the observed leakage of alkaline phosphatase and β‑galactosidase from cells. GEP was demonstrated to perturb the metabolism of the microorganisms, especially the tricarboxylic acid cycle, as indicated by the reduced intracellular activity of succinate dehydrogenase, malate dehydrogenase and ATPases, including the Na+/K+‑ATPase and the Ca2+‑ATPase. In addition, GEP caused the leakage of the genetic material of the bacteria and the fungus, as indicated by the increased OD260. The results of the present study indicated that GEP may exert its antimicrobial activity by damaging cell walls and membranes, causing the leakage of genetic material, and by perturbing cellular metabolism.

Introduction

Fungal and bacterial infections cause serious problems in medicine, the environment and the food industry. Candida albicans causes vaginal candidiasis, which affects 75 million women every year (1). Staphylococcus aureus, the most common pathogen in human purulent infections, is responsible for food poisoning, osteomyelitis, and chronic and recurrent infections that are difficult to prevent using antibiotics (2). Antibiotic resistance in bacteria, such as S. aureus and Pseudomonas aeruginosa, is becoming increasingly prevalent and serious (3). Escherichia coli can cause gastrointestinal or urethral tract infections in humans and animals under certain conditions. A range of existing biocides, including antibiotics (4), metal particles (silver or tin), titanium oxide (5) and quaternary ammonium salts (6), have been banned owing to their toxicity (7). Researchers are therefore investigating natural broad-spectrum antibacterial agents that may be used to reduce the problem of drug resistance (810). Plant-derived compounds, including tannins, flavonoids, alkaloids and peptides have been reported to exhibit therapeutic action against bacterial infection (1113), indicating their utility as antibacterial substances. Antibacterial drugs are classified according to four mechanism of action: i) interference with cell wall synthesis; ii) causing damage to the plasma membrane; iii) inhibiting bacterial protein synthesis; and iv) affecting nucleic acid metabolism.

The rhizome of the orchid Gastrodia elata Blume, has been used medicinally in Asian countries for >2,000 years (14,15) owing to its various pharmacological actions, including anti-epilepsy (16), anti-convulsion (15), anti-inflammation (17,18) and anti-tetanus (16). Among its active ingredients, the polypeptides have received wide attention. G. elata protein extracts have been reported to have significant antifungal activity and antibacterial activity against gram-positive bacteria, and exhibits little hemolytic activity on rabbit red blood cells (19). In our previous study, the antimicrobial activities of polypeptide-enriched G. elata extracts (GEP) were demonstrated using the agar diffusion method (20) and its effects on C. albicans were confirmed in a mouse model of vulvovaginal candidiasis. However, the antimicrobial mechanism of action of GEP has, to the best of our knowledge, not yet been reported.

The present study aimed to investigate the possible mechanism of GEP-mediated antimicrobial efficacy on C. albicans, E. coli, S. aureus and P. aeruginosa by determining its effect on cell walls, cell membranes and the levels of intracellular and extracellular proteins. Data from the present study suggested that the antimicrobial effects of GEP may be related to the modulation of membrane integrity and intracellular energy metabolism.

Materials and methods

Preparation of GEP and molar mass distribution analysis

In the Changbai Mountain region (Jilin, China), G. elata has been planted artificially, and is an economic species. In the present study, the cultured G. elata rhizomes were purchased from Taobao and stored at room temperature. The rhizomes of G. elata were crushed and extracted for 2 h in saline at 80°C, the resulting solution was then filtered. Papain (Shanghai Yuanye Biological Technology Co., Ltd.) was added to the collected filtrate to give a final concentration of 0.32 g/liter. The resulting solution was heated at 60°C for 30 min (to trigger papain-mediated hydrolysis) and then heated in a water bath at 100°C for a further 40 min (to inactivate the papain). Subsequently, the solution was passed through a 10 kDa ultrafiltration membrane (Sartocon 2 PES; Sartorius AG), and the resulting filtrate was concentrated by rotary evaporation at 60°C with a rotation speed of 10 rpm until the extract was determined to have a concentration of 22.5 g/l using the bicinchoninic acid (BCA) method.

The molar mass distribution of GEP was determined using gel permeation chromatography (Wyatt Technology) equipped with two serial OHpak polymer matrix phase columns (OHpak SB-806 and 803; 8.0×300 mm; Shodex). The samples were eluted using a 0.02% aqueous solution of NaN3, at a flow-rate of 1 ml/min at 40°C. Calculations of the average molar masses [number average molar mass (Mn), weight-average molar mass (Mw), z-average molar mass (Mz)] and dispersity index (Mw/Mn) were calculated using Omi SEC v4.7 software (Malvern Panalytical).

Strains and culture condition

In total, four microbial strains, C. albicans (cat. no. ATCC 10231), E. coli (cat. no. 8099), S. aureus (cat. no. ATCC 6538) and P. aeruginosa (cat. no. ATCC 27853) were supplied by the Guangdong Microbial Culture Center. The bacterial strains were cultured in lysogeny broth (10 g/l peptone, 5 g/l yeast extract and 10 g/l NaCl) at 37°C with shaking at 150 rpm for 20 h. C. albicans was cultured in Sabouraud Dextrose Broth (Qingdao Hope Bio-Technology Co., Ltd.) at 28°C with shaking at 150 rpm for 20 h.

Determination of the minimum inhibitory concentration (MIC)

After culturing for 20 h, suspensions with a final concentration of 1×106 colony-forming units (CFU)/ml were collected. For each microbial strain, 100 µl aliquots of prepared suspension were added to 96-well plates and 100 µl aliquots containing different concentrations of GEP diluted in PBS were added to the wells (1.127, 2.254, 4.508, 6.762, 9.016, 11.270 and 13.524 mg/ml of GEP for C. albicans and 1.803, 2.254, 2.705, 3.156, 3.606, 4.057 and 4.508 mg/ml of GEP for E. coli, S. aureus and P. aeruginosa); 100 µl of PBS was added to the negative control wells and 100 µl of 75% ethanol (analytical pure) was added to the positive control wells. The plates were cultured for 24 h at 37°C (bacterial strains) or 28°C (C. albicans), and 20 µl of 0.2% triphenyltetrazolium chloride (Sigma-Aldrich; Merck KGaA) was added to the bacterial suspensions and 20 µl of 5 mg/ml MTT was added to the fungal suspensions, as previously described (21,22). The plates were incubated for a further 4 h in darkness. MIC was defined as the minimum concentration of GEP that completely inhibited cell viability, as shown by the absence of formazan crystal formation. After observation, formazan crystals were dissolved using DMSO and were measured at 490 nm for the three bacterial strains and 570 nm for C. albicans. The same results were obtained from analysis under a microscope.

Construction of growth curves

In total, 20 ml of bacterial or fungal suspensions (1×106 CFU/ml) was mixed with 20 ml of 2X MIC GEP (1X MIC treated group) or PBS (Control group), and cultured at the aforementioned temperatures. The optical density at 600 nm was determined using 1 ml of mixture using a Synergy HT Multi-detection microplate reader (Omega Bio-Tek, Inc.) at 0, 1, 2, 4, 6, 8 and 10 h for E. coli and P. aeruginosa, and 0, 1, 2, 4, 6, 8, 12, 16 and 20 h for C. albicans and S. aureus.

Scanning electron microscopy (SEM) analysis

The bacterial and fungal suspensions at a final density of 5×105 CFU/ml mixed with GEP (1X MIC treatment group) or an equal volume of PBS (Control group) were incubated for 16 h at 37°C (bacterial strains) or 28°C (C. albicans). After centrifugation at 604 × g at room temperature for 5 min and washing with PBS three times, the collected precipitates were fixed with 2.5% (v/v) glutaraldehyde for 2 h at 4°C, dehydrated by an ascending series of ethanol washes (30–100%), freeze-dried and gold-sputtered. Samples were observed by SEM using a Hitachi X650 (Hitachi, Ltd.).

Determination of optical density (OD) at 260 nm to detect DNA and RNA

The bacterial and fungal suspensions (final density, 5×105 CFU/ml) mixed with GEP (1X MIC treatment group) or PBS (Control group) were incubated for 0 or 2 h. At each time point, 1 ml of mixture was removed, centrifuged at 9,660 × g at room temperature for 10 min and the resulting supernatant passed through a microporous filter (0.22 µm; EMD Millipore). The optical density of the resulting supernatant was measured at 260 nm using a microplate reader (23).

Determination of the activities of extracellular enzymes

In the preliminary experiments to determine extracellular alkaline phosphatase (AKP) activity, samples were collected from the supernatants of each strain after 1, 2, 4, 8 and 10 h 1X MIC GEP or PBS Control treatment. AKP activity was detected using an AKP assay kit (cat. no. A059-1; Nanjing Jiancheng Bioengineering Institute). According to the preliminary results (Fig. S1), a 2 h incubation was selected for the subsequent experiments.

In the preliminary experiments, the extracellular β-galactosidase activity was detected in the cultured medium after 1, 2, 4, 8 and 10 h 1X MIC GEP or PBS Control treatment. According the preliminary results (Fig. S2), a 4 h incubation was selected for the subsequent experiments. Samples (100 µl) were added to 400 µl 0.05 mol/l O-nitrophenyl-β-D-galactopyranoside (ONPG; Sigma-Aldrich; Merck KGaA) and the mixture was incubated in a water bath at 37°C for 40 min. The resulting mixture was immediately combined with 500 µl 0.5 mol/l sodium bicarbonate and the optical density at 420 nm was measured using a microplate reader. The formula for the calculation of β-galactosidase activity is as follows (24,25): β-Galactosidase activity (U/ml)=(OD420 × A)/(B × C × 0.0045) where A represents the volume of the system (ml), B represents the incubation time (min), C represents the sample volume (ml) and 0.0045 represents the molar extinction coefficient of ONPG (ml/nmol).

Determination of the activities of intracellular enzymes

After incubation for 10 h, the treated bacteria and fungi were collected by centrifugation at 604 × g at room temperature for 5 min and washed with PBS. Intracellular proteins were extracted using a bacterial protein extraction kit (cat. no. BB-3123; Best Science) or yeast total protein extraction kit (cat. no. BB-3125; Best Bio Science). The concentration of the protein samples was determined using the BCA method.

The activities of intracellular malate dehydrogenase (MDH; cat. no. A021-2), succinate dehydrogenase (SDH; cat. no. A022), ATPase (cat. no. A095-1), Na+/K+-ATPase (cat. no. A070-2) and Ca2+-ATPase (cat. no. A070-4) were determined according to the instructions for the commercial kits (all from Nanjing Jiancheng Bioengineering Institute).

Statistical analysis

All data are presented as the mean ± SD and represent six independent experiments; each experiment was performed in triplicate. Statistical significance was determined using two-tailed Student's t-tests using SPSS Statistics v24 (IBM Corp.) for Windows. P>0.05 was considered to indicate a statistically significant difference.

Results

Molar mass distribution of GEP

Mw/Mn is defined as the polydispersity index, representing the dispersion of the molecular weight of GEP. The smaller the polydispersity index value, the more uniform in molar mass the sample is. The Mw/Mn of GEP was determined to be 4.820, which suggested that the molecular weight of GEP is moderately uniform (Table I).

Table I.

The molar mass distribution of GEP.

Table I.

The molar mass distribution of GEP.

ParameterGEP
Mw/Mn4.820
Mn (g/mol) 1.102×104
Mw (g/mol) 5.310×104
Mz (g/mol) 3.142×105

[i] GEP, polypeptide-enriched Gastrodia elata extracts; Mn, number average molar mass; Mw, weight-average molar mass; Mz, z-average molar mass.

MICs of GEP against the four microbial strains

GEP had the highest MIC value for C. albicans (5.635 mg/ml) and the lowest MIC value for S. aureus (1.127 mg/ml) (Table II). The MIC values of GEP against E. coli and P. aeruginosa were 1.803 and 1.352 mg/ml, respectively. These data suggested that gram-negative bacteria (E. coli, P. aeruginosa) and gram-positive bacteria (S. aureus) are more sensitive to GEP than fungi (C. albicans).

Table II.

MICs of polypeptide-enriched Gastrodia elata extracts against four microbial strains.

Table II.

MICs of polypeptide-enriched Gastrodia elata extracts against four microbial strains.

Microbial strainMIC (mg/ml)
Candida albicans5.635
Escherichia coli1.803
Staphylococcus aureus1.127
Pseudomonas aeruginosa1.352

[i] MIC, minimum inhibitory concentration.

Effects of GEP on the growth of the four microbial strains

Growth curves were constructed from 0 to 10 or 20 h for the different strains to investigate the effects of GEP at 1X MIC on antimicrobial properties over time (26). Compared with the respective PBS-treated Control groups, the number of cells was significantly lower following treatment with GEP (1X MIC), which suggested that GEP inhibits the growth of C. albicans, E. coli, S. aureus and P. aeruginosa (Fig. 1); GEP almost completely inhibited the growth of E. coli (Fig. 1B) and S. aureus (Fig. 1C).

Effects of GEP on cell wall integrity

To investigate the morphology and the integrity of the cell wall of the four microbial strains, SEM was performed and the activity of AKP was determined. PBS-treated C. albicans and S. aureus cells had a smooth and plump sphere shape, whereas following 16 h of GEP treatment vesication or the formation of irregular protruding structures were observed on the cell surface (Fig. 2). For E. coli and P. aeruginosa, compared with the control group, cell-surface damage, such as uneven spots and curves, was apparent in the GEP-treated groups (Fig. 2). Some visible cell debris was observed in the GEP-treated E. coli and S. aureus (Fig. 2).

AKP, a phosphatase found between the cell wall and cell membrane, serves as an index for cell wall permeability (27). Compared with the Control group, GEP (1X MIC) treatment for 2 h caused a 43.9-fold (P<0.001), 10.0-fold (P<0.001), 4.4-fold (P<0.001) and 8.8-fold (P<0.001) increase in the extracellular AKP activity for C. albicans, E. coli, S. aureus and P. aeruginosa, respectively (Fig. 3), which suggested that GEP treatment resulted in damage and disruption to the cell wall.

Effects of GEP on cell membrane permeability

β-Galactosidase is an intracellular enzyme that leaks from cells following damage to the cell membrane; therefore, β-galactosidase can be used as an indicator of cell membrane integrity (25). Compared with the Control group, treatment with GEP (1X MIC) for 4 h resulted in a 28.3 (P<0.001), 7.6 (P<0.001), 8.1 (P<0.01) and 7.1% (P<0.01) increase in extracellular β-galactosidase activity in the samples of C. albicans, E. coli, S. aureus and P. aeruginosa, respectively (Fig. 4A).

Ion pumps, such as the Na+/K+-ATPase and the Ca2+-ATPase, are important membrane proteins that regulate cell membrane permeability (28). Compared with the Control group, treatment with GEP (1X MIC) resulted in a decrease in the activities of Na+/K+-ATPase (Fig. 4B) and Ca2+-ATPase (Fig. 4C) of 88.3 (P<0.001) and 73.5% (P<0.001), respectively, for C. albicans; 16.9 (P<0.001) and 65.5% (P<0.001), respectively, for E. coli; 64.7 (P<0.001) and 36.9% (P<0.001), respectively, for S. aureus; and 51.3 (P<0.001) and 22.7% (P<0.001) for P. aeruginosa. These findings suggested that GEP damaged the cell membranes of these microbial strains.

Effects of GEP on the leakage of genetic material

A high OD260 may be detected following the leakage of nucleotides (25). Compared with the Control group, GEP (1X MIC) treatment caused a 590% (P<0.001) increase in the OD260 between 0 and 2 h in S. aureus and a 530% (P<0.001) increase for P. aeruginosa (Fig. 5); a 100 (P<0.001) and 80% (P<0.01) increase in the OD was observed for C. albicans and E. coli, respectively. These results indicated that GEP may cause leakage of genetic material from these microorganisms.

Effects of GEP on intracellular enzyme activity

SDH, MDH and ATPases serve important roles in microbial energy metabolism. ATPases hydrolyze ATP and provide energy to drive other chemical reaction in the cell (29). Compared with the Control group, GEP (1X MIC) treatment reduced the activity of ATPases by 100.0 (P<0.001), 39.4 (P<0.01), 93.0 (P<0.001) and 87.1% (P<0.001) in C. albicans, E. coli, S. aureus and P. aeruginosa, respectively (Fig. 6A).

MDH affects biosynthesis through its coenzyme NADP+ (29); SDH is an important marker of the health of microbial energy metabolism. Compared with the Control group, GEP (1X MIC) treatment reduced the activity of MDH by 78.4 (P<0.001), 76.6 (P<0.001), 89.4 (P<0.001) and 50.2% (P<0.01) in C. albicans, E. coli, S. aureus and P. aeruginosa, respectively (Fig. 6B). GEP treatment suppressed the activity of SDH by 21.1 (P<0.05), 48.6 (P<0.001), 25.1 (P<0.001) and 70.7% (P<0.001) in C. albicans, E. coli, S. aureus and P. aeruginosa, respectively (Fig. 6C). These data suggested that GEP treatment disturbed energy metabolism in these microorganisms.

Discussion

The antimicrobial activity of GEP against fungi and gram-positive bacteria has been reported previously (19,20). Our previous study demonstrated the antimicrobial activities of GEP using the agar diffusion method (20) and are now investigating the therapeutic efficacy of GEP against vulvovaginal candidiasis in mice (30). In the present study, it was demonstrated that the effects of GEP may derive from the damage caused to cell walls and membranes, triggering the leakage of genetic material and disrupting cellular metabolism.

The SEM results demonstrated that GEP damaged cell walls and membranes, leading to changes in cell morphology and cell death These changes may result in the loss of intracellular material (31). Various antibacterial agents act primarily on the cell membrane (32), which is a selectively permeable barrier that enables the normal growth of bacteria (23). Leakage of intracellular substances serves as an indicator of membrane integrity. AKP is present between the cell wall and the cell membrane (33,34). Damage to the cell wall increases cell permeability, leading to the leakage of AKP (27). The enhanced extracellular AKP levels within 2 h of treatment with GEP are consistent with the SEM results, and further suggested that GEP damaged cell walls and membranes.

β-Galactosidase is an important microbial enzyme that hydrolyzes lactose into galactose and glucose to produce energy, and provide a source of carbon (35). In the present study, it was demonstrated that GEP may regulate energy metabolism in bacteria and fungi. In addition to enhancing the extracellular activities of β-galactosidase, GEP directly suppressed the intracellular activity levels of SDH and MDH in the four microbial strains. The tricarboxylic acid (TCA) cycle is the metabolic pathway linking the three major classes of nutrients (sugars, lipids and amino acids) and the main energy production mode of the four microbial strains examined. MDH catalyzes the reversible conversion of malic acid and oxaloacetic acid in the last step of the TCA cycle, influencing ATP production (36,37). SDH provides electrons for the respiratory chain. Decreased SDH activity inhibits mitochondrial electron transport and oxidative respiration, resulting in ATP consumption, thus hindering the TCA cycle and energy metabolism (37,38).

ATPases hydrolyzes ATP to release energy (23). As previously reported, the inhibition of the ATPase activity of Ca2+-ATPase and Na+/K+-ATPase leads to an overload of intracellular Ca2+ and Na+, the inhibition of energy metabolism, a reduction in ATP production and an imbalance of osmotic pressure, which causes cell swelling and apoptosis (38). The data from the present study suggested that these changes in energy metabolism may be one of the reasons for the antimicrobial efficacy of GEP.

In the present study, the possible mechanisms underlying the antimicrobial activities of GEP were investigated, and the results indicated that GEP may disrupt energy metabolism, especially the TCA cycle, and may also be able to damage microbial cell walls and membranes, causing the leakage of genetic material.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was funded by The Special Projects of the Cooperation between Jilin University and Jilin Province (grant no. SXGJXX2017-1) and The Industrial Technology Research and Development Projects from Development and Reform Commission of Jilin Province (grant no. 2019C050-8).

Availability of data and materials

All data generated and analyzed during this study are included in this published article.

Authors' contributions

DW and HB designed the experiments. FK, XC, SZ, RW, XZ and YM performed the experiments. FK, XC, SZ and RW contributed to data analysis and proofreading. DW, FK and XC wrote the paper. DW and HB revised the paper.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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November-2019
Volume 20 Issue 5

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Copy and paste a formatted citation
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Spandidos Publications style
Kong F, Cai X, Zhai S, Wang R, Zheng X, Ma Y, Bi H and Wang D: Possible mechanisms of the antimicrobial effects of polypeptide‑enriched Gastrodia elata Blume extracts. Mol Med Rep 20: 4723-4730, 2019
APA
Kong, F., Cai, X., Zhai, S., Wang, R., Zheng, X., Ma, Y. ... Wang, D. (2019). Possible mechanisms of the antimicrobial effects of polypeptide‑enriched Gastrodia elata Blume extracts. Molecular Medicine Reports, 20, 4723-4730. https://doi.org/10.3892/mmr.2019.10706
MLA
Kong, F., Cai, X., Zhai, S., Wang, R., Zheng, X., Ma, Y., Bi, H., Wang, D."Possible mechanisms of the antimicrobial effects of polypeptide‑enriched Gastrodia elata Blume extracts". Molecular Medicine Reports 20.5 (2019): 4723-4730.
Chicago
Kong, F., Cai, X., Zhai, S., Wang, R., Zheng, X., Ma, Y., Bi, H., Wang, D."Possible mechanisms of the antimicrobial effects of polypeptide‑enriched Gastrodia elata Blume extracts". Molecular Medicine Reports 20, no. 5 (2019): 4723-4730. https://doi.org/10.3892/mmr.2019.10706