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Research article

Molecular and Cellular Microbiology (MCM)  |  Clinical Microbiology and Biomedical Sciences

Paeonia lactiflora Inhibits Cell Wall Synthesis and Triggers Membrane Depolarization in Candida albicans

Heung-Shick Lee 1 and Younhee Kim 2*

1Department of Biotechnology and Bioinformatics, Korea University, Sejongsi 30019, Republic of Korea, 2Department of Korean Medicine, Semyung University, Jecheon 27136, Republic of Korea

Received: November 24, 2016; Accepted: January 13, 2017

J. Microbiol. Biotechnol. 2017; 27(2): 395-404

Published February 28, 2017 https://doi.org/10.4014/jmb.1611.11064

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Fungal cell walls and cell membranes are the main targets of antifungals. In this study, we report on the antifungal activity of an ethanol extract from Paeonia lactiflora against Candida albicans, showing that the antifungal activity is associated with the synergistic actions of preventing cell wall synthesis, enabling membrane depolarization, and compromising permeability. First, it was shown that the ethanol extract from P. lactiflora was involved in damaging the integrity of cell walls in C. albicans. In isotonic media, cell bursts of C. albicans by the P. lactiflora ethanol extract could be restored, and the minimum inhibitory concentration (MIC) of the P. lactiflora ethanol extract against C. albicans cells increased 4-fold. In addition, synthesis of (1,3)-β-D-glucan polymer was inhibited by 87% and 83% following treatment of C. albicans microsomes with the P. lactiflora ethanol extract at their 1× MIC and 2× MIC, respectively. Second, the ethanol extract from P. lactiflora influenced the function of C. albicans cell membranes. C. albicans cells treated with the P. lactiflora ethanol extract formed red aggregates by staining with a membrane-impermeable dye, propidium iodide. Membrane depolarization manifested as increased fluorescence intensity by staining P. lactiflora-treated C. albicans cells with a membrane-potential marker, DiBAC4(3) ((bis-1,3-dibutylbarbituric acid) trimethine oxonol). Membrane permeability was assessed by crystal violet assay, and C. albicans cells treated with the P. lactiflora ethanol extract exhibited significant uptake of crystal violet in a concentration-dependent manner. The findings suggest that P. lactiflora ethanol extract is a viable and effective candidate for the development of new antifungal agents to treat Candida-associated diseases.

Keywords

Antifungal, Candida albicans, cell wall, membrane permeability, membrane potential, Paeonia lactiflora

References

  1. Pfaller MA, Pappas PG, Wingard JR. 2006. Invasive fungal pathogens: current epidemiological trends. Clin. Infect. Dis. 43: S3-S14.
    CrossRef
  2. Eliopoulos GM, Perea S, Patterson TF. 2002. Antifungal resistance in pathogenic fungi. Clin. Infect. Dis. 35: 1073-1080.
    Pubmed CrossRef
  3. Jenkinson HF, Douglas LJ. 2002. Interactions between Candida species and bacteria in mixed infections. Brogden KA, Guthmiller JM (eds.). Polymicrobial Diseases. ASM Press, Washington, DC.
    CrossRef
  4. Lortholary O, Dupont B. 1997. Antifungal prophylaxis during neutropenia and immunodeficiency. Clin. Microbiol. Rev. 10: 477-504.
    Pubmed PMC
  5. Vandeputte P, Ferrari S, Coste AT. 2011. Antifungal resistance and new strategies to control fungal infections. Int. J. Microbiol. 2012: 1-26.
    Pubmed PMC CrossRef
  6. White TC, Marr KA, Bowden RA. 1998. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin. Microbiol. Rev. 11: 382-402.
    Pubmed PMC
  7. Gray KC, Palacios DS, Dailey I, Endo MM, Uno BE, Wilcock BC, Burke MD. 2012. Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. USA 109:2234-2239.
    Pubmed PMC CrossRef
  8. Carrillo-Muñoz AJ, Giusiano G, Ezkurra PA, Quindós G. 2006. Antifungal agents: mode of action in yeast cells. Rev. Esp. Quimioter. 19: 130-139.
  9. Kurtz M, Douglas C. 1997. Lipopeptide inhibitors of fungal glucan synthase. J. Med. Vet. Mycol. 35: 79-86.
    Pubmed CrossRef
  10. Vermes A, Guchelaar HJ, Dankert J. 2000. Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J. Antimicrob. Chemother. 46:171-179.
    Pubmed CrossRef
  11. Carson CF, Mee BJ, Riley TV. 2002. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46: 1914-1920.
    Pubmed PMC CrossRef
  12. Shapiro RS, Robbins N, Cowen LE. 2011. Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol. Mol. Biol. Rev. 75: 213-267.
    Pubmed PMC CrossRef
  13. Cowan MM. 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12: 564-582.
    Pubmed PMC
  14. Kim CM, Shin MK, Ahn DK, Lee KS (eds.). 1998. The Encyclopedia of Oriental Herbal Medicine (Korean version), 1st Ed. Jeongdam Press, Seoul, Korea.
  15. Zhang W, Dai SM. 2012. Mechanisms involved in the therapeutic effects of Paeonia lactiflora Pallas in rheumatoid arthritis. Int. Immunopharmacol. 14: 27-31.
    Pubmed CrossRef
  16. Clinical and Laboratory Standards Institute. 2008. M27-A3. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. 3rd Ed. Clinical and Laboratory Standards Institute, Wayne, PA.
  17. Liu M, Seidel V, Katerere DR, Gray AI. 2007. Colorimetric broth microdilution method for the antifungal screening of plant extracts against yeast. Methods 42: 325-329.
    Pubmed CrossRef
  18. Frost DJ, Brandt KD, Cugier D, Goldman R. 1995. A wholecell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J. Antibiot. 48: 306-310.
    Pubmed CrossRef
  19. Shedletzky E, Unger C, Delmer DP. 1997. A microtiter-based fluorescence assay for (1,3)-β-glucan synthases. Anal. Biochem. 249: 88-93.
    Pubmed CrossRef
  20. Lee HS, Kim Y. 2016. Antifungal activity of Salvia miltiorrhiza against Candida albicans is associated with the alteration of membrane permeability and (1,3)-β-D-glucan synthase activity. J. Microbiol. Biotechnol. 26: 610-617.
    Pubmed CrossRef
  21. Vaara M, Vaara T. 1981. Outer membrane permeability barrier disruption by polymixin in polymixin-susceptible and resistant Salmonella typhimurium. Antimicrob. Agents Chemother. 19: 578-583.
    Pubmed PMC CrossRef
  22. Lee HS, Kim Y. 2014. Antifungal activity of Rheum undulatum on Candida albicans by the changes in membrane permeability. Korean J. Microbiol. 50: 360-367.
    CrossRef
  23. Halder S, Yadav KK, Sarkar R, Mukherjee S, Saha P, Haldar S, et al. 2015. Alteration of zeta potential and membrane permeability in bacteria: a study with cationic agents. SpringerPlus 4: 672.
    Pubmed PMC CrossRef
  24. Choi IH, Kim Y, Lee DN, Kim HJ. 2005. Antifungal effects of Cinamon Ramulus, Pulsatillae Radix, Dictamni Radicis Cortex, Paeonia Radix, Arecae Semen, Artemisiae Capillaries Herba against Candida albicans. Korean J. Orient. Physiol. Pathol. 19: 690-695.
  25. Onishi J, Meinz M, Thompson J, Curotto J, Dreikorn S, Rosenbach M, et al. 2000. Discovery of novel antifungal (1,3)-β-D-glucan synthase inhibitors. Antimicrob. Agents Chemother. 44: 368-377.
    Pubmed PMC CrossRef
  26. Chandra J, Patel JD, Li J, Zhou G, Mukherjee PK, McCormick TS, et al. 2005. Modification of surface properties of biomaterials influences the ability of Candida albicans to form biofilms. Appl. Environ. Microbiol. 71: 8795-8801.
    Pubmed PMC CrossRef
  27. Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. 1986. The rate of killing of Escherichia coli by β-lactam antibiotics is strictly proportional to the rate of bacterial growth. J. Gen. Microbiol. 132: 1297-1304.
    CrossRef
  28. Ehara M, Noguchi T, Ueda K. 1996. Uptake of neutral red by the vacuoles of a green alga, Micrasterias pinnatifida. Plant Cell Physiol. 37: 734-741.
    CrossRef
  29. Viarengo A, Lowe D, Bolognesi C, Fabbri E, Koehler A. 2007. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms. Comp. Biochem. Physiol. C 146: 281-300.
    CrossRef
  30. Li SC, Kane PM. 2009. The yeast lysosome-like vacuole:endpoint and crossroads. Biochim. Biophys. Acta 1793: 650-663.
    Pubmed PMC CrossRef
  31. Wilson HA, Chused TM. 1985. Lymphocyte membrane potential and Ca2+ sensitivity potassium channels described by oxonol dye fluorescence measurements. J. Cell. Physiol. 125: 72-81.
    Pubmed CrossRef
  32. Volkov V. 2 015. Q uantitative description o f ion t ransport via plasma membrane of yeast and small cells. Front. Plant Sci. 6: 1-22.
  33. Devi KP, Nisha SA, Sakthivel R, Pandian SK. 2010. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J. Ethnopharmacol. 130: 107-115.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(2): 395-404

Published online February 28, 2017 https://doi.org/10.4014/jmb.1611.11064

Copyright © The Korean Society for Microbiology and Biotechnology.

Paeonia lactiflora Inhibits Cell Wall Synthesis and Triggers Membrane Depolarization in Candida albicans

Heung-Shick Lee 1 and Younhee Kim 2*

1Department of Biotechnology and Bioinformatics, Korea University, Sejongsi 30019, Republic of Korea, 2Department of Korean Medicine, Semyung University, Jecheon 27136, Republic of Korea

Received: November 24, 2016; Accepted: January 13, 2017

Abstract

Fungal cell walls and cell membranes are the main targets of antifungals. In this study, we
report on the antifungal activity of an ethanol extract from Paeonia lactiflora against Candida
albicans, showing that the antifungal activity is associated with the synergistic actions of
preventing cell wall synthesis, enabling membrane depolarization, and compromising
permeability. First, it was shown that the ethanol extract from P. lactiflora was involved in
damaging the integrity of cell walls in C. albicans. In isotonic media, cell bursts of C. albicans by
the P. lactiflora ethanol extract could be restored, and the minimum inhibitory concentration
(MIC) of the P. lactiflora ethanol extract against C. albicans cells increased 4-fold. In addition,
synthesis of (1,3)-β-D-glucan polymer was inhibited by 87% and 83% following treatment of C.
albicans microsomes with the P. lactiflora ethanol extract at their 1× MIC and 2× MIC,
respectively. Second, the ethanol extract from P. lactiflora influenced the function of C. albicans
cell membranes. C. albicans cells treated with the P. lactiflora ethanol extract formed red
aggregates by staining with a membrane-impermeable dye, propidium iodide. Membrane
depolarization manifested as increased fluorescence intensity by staining P. lactiflora-treated
C. albicans cells with a membrane-potential marker, DiBAC4(3) ((bis-1,3-dibutylbarbituric acid)
trimethine oxonol). Membrane permeability was assessed by crystal violet assay, and C.
albicans cells treated with the P. lactiflora ethanol extract exhibited significant uptake of crystal
violet in a concentration-dependent manner. The findings suggest that P. lactiflora ethanol
extract is a viable and effective candidate for the development of new antifungal agents to
treat Candida-associated diseases.

Keywords: Antifungal, Candida albicans, cell wall, membrane permeability, membrane potential, Paeonia lactiflora

References

  1. Pfaller MA, Pappas PG, Wingard JR. 2006. Invasive fungal pathogens: current epidemiological trends. Clin. Infect. Dis. 43: S3-S14.
    CrossRef
  2. Eliopoulos GM, Perea S, Patterson TF. 2002. Antifungal resistance in pathogenic fungi. Clin. Infect. Dis. 35: 1073-1080.
    Pubmed CrossRef
  3. Jenkinson HF, Douglas LJ. 2002. Interactions between Candida species and bacteria in mixed infections. Brogden KA, Guthmiller JM (eds.). Polymicrobial Diseases. ASM Press, Washington, DC.
    CrossRef
  4. Lortholary O, Dupont B. 1997. Antifungal prophylaxis during neutropenia and immunodeficiency. Clin. Microbiol. Rev. 10: 477-504.
    Pubmed KoreaMed
  5. Vandeputte P, Ferrari S, Coste AT. 2011. Antifungal resistance and new strategies to control fungal infections. Int. J. Microbiol. 2012: 1-26.
    Pubmed KoreaMed CrossRef
  6. White TC, Marr KA, Bowden RA. 1998. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin. Microbiol. Rev. 11: 382-402.
    Pubmed KoreaMed
  7. Gray KC, Palacios DS, Dailey I, Endo MM, Uno BE, Wilcock BC, Burke MD. 2012. Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. USA 109:2234-2239.
    Pubmed KoreaMed CrossRef
  8. Carrillo-Muñoz AJ, Giusiano G, Ezkurra PA, Quindós G. 2006. Antifungal agents: mode of action in yeast cells. Rev. Esp. Quimioter. 19: 130-139.
  9. Kurtz M, Douglas C. 1997. Lipopeptide inhibitors of fungal glucan synthase. J. Med. Vet. Mycol. 35: 79-86.
    Pubmed CrossRef
  10. Vermes A, Guchelaar HJ, Dankert J. 2000. Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J. Antimicrob. Chemother. 46:171-179.
    Pubmed CrossRef
  11. Carson CF, Mee BJ, Riley TV. 2002. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46: 1914-1920.
    Pubmed KoreaMed CrossRef
  12. Shapiro RS, Robbins N, Cowen LE. 2011. Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol. Mol. Biol. Rev. 75: 213-267.
    Pubmed KoreaMed CrossRef
  13. Cowan MM. 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12: 564-582.
    Pubmed KoreaMed
  14. Kim CM, Shin MK, Ahn DK, Lee KS (eds.). 1998. The Encyclopedia of Oriental Herbal Medicine (Korean version), 1st Ed. Jeongdam Press, Seoul, Korea.
  15. Zhang W, Dai SM. 2012. Mechanisms involved in the therapeutic effects of Paeonia lactiflora Pallas in rheumatoid arthritis. Int. Immunopharmacol. 14: 27-31.
    Pubmed CrossRef
  16. Clinical and Laboratory Standards Institute. 2008. M27-A3. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. 3rd Ed. Clinical and Laboratory Standards Institute, Wayne, PA.
  17. Liu M, Seidel V, Katerere DR, Gray AI. 2007. Colorimetric broth microdilution method for the antifungal screening of plant extracts against yeast. Methods 42: 325-329.
    Pubmed CrossRef
  18. Frost DJ, Brandt KD, Cugier D, Goldman R. 1995. A wholecell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J. Antibiot. 48: 306-310.
    Pubmed CrossRef
  19. Shedletzky E, Unger C, Delmer DP. 1997. A microtiter-based fluorescence assay for (1,3)-β-glucan synthases. Anal. Biochem. 249: 88-93.
    Pubmed CrossRef
  20. Lee HS, Kim Y. 2016. Antifungal activity of Salvia miltiorrhiza against Candida albicans is associated with the alteration of membrane permeability and (1,3)-β-D-glucan synthase activity. J. Microbiol. Biotechnol. 26: 610-617.
    Pubmed CrossRef
  21. Vaara M, Vaara T. 1981. Outer membrane permeability barrier disruption by polymixin in polymixin-susceptible and resistant Salmonella typhimurium. Antimicrob. Agents Chemother. 19: 578-583.
    Pubmed KoreaMed CrossRef
  22. Lee HS, Kim Y. 2014. Antifungal activity of Rheum undulatum on Candida albicans by the changes in membrane permeability. Korean J. Microbiol. 50: 360-367.
    CrossRef
  23. Halder S, Yadav KK, Sarkar R, Mukherjee S, Saha P, Haldar S, et al. 2015. Alteration of zeta potential and membrane permeability in bacteria: a study with cationic agents. SpringerPlus 4: 672.
    Pubmed KoreaMed CrossRef
  24. Choi IH, Kim Y, Lee DN, Kim HJ. 2005. Antifungal effects of Cinamon Ramulus, Pulsatillae Radix, Dictamni Radicis Cortex, Paeonia Radix, Arecae Semen, Artemisiae Capillaries Herba against Candida albicans. Korean J. Orient. Physiol. Pathol. 19: 690-695.
  25. Onishi J, Meinz M, Thompson J, Curotto J, Dreikorn S, Rosenbach M, et al. 2000. Discovery of novel antifungal (1,3)-β-D-glucan synthase inhibitors. Antimicrob. Agents Chemother. 44: 368-377.
    Pubmed KoreaMed CrossRef
  26. Chandra J, Patel JD, Li J, Zhou G, Mukherjee PK, McCormick TS, et al. 2005. Modification of surface properties of biomaterials influences the ability of Candida albicans to form biofilms. Appl. Environ. Microbiol. 71: 8795-8801.
    Pubmed KoreaMed CrossRef
  27. Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. 1986. The rate of killing of Escherichia coli by β-lactam antibiotics is strictly proportional to the rate of bacterial growth. J. Gen. Microbiol. 132: 1297-1304.
    CrossRef
  28. Ehara M, Noguchi T, Ueda K. 1996. Uptake of neutral red by the vacuoles of a green alga, Micrasterias pinnatifida. Plant Cell Physiol. 37: 734-741.
    CrossRef
  29. Viarengo A, Lowe D, Bolognesi C, Fabbri E, Koehler A. 2007. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms. Comp. Biochem. Physiol. C 146: 281-300.
    CrossRef
  30. Li SC, Kane PM. 2009. The yeast lysosome-like vacuole:endpoint and crossroads. Biochim. Biophys. Acta 1793: 650-663.
    Pubmed KoreaMed CrossRef
  31. Wilson HA, Chused TM. 1985. Lymphocyte membrane potential and Ca2+ sensitivity potassium channels described by oxonol dye fluorescence measurements. J. Cell. Physiol. 125: 72-81.
    Pubmed CrossRef
  32. Volkov V. 2 015. Q uantitative description o f ion t ransport via plasma membrane of yeast and small cells. Front. Plant Sci. 6: 1-22.
  33. Devi KP, Nisha SA, Sakthivel R, Pandian SK. 2010. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J. Ethnopharmacol. 130: 107-115.
    Pubmed CrossRef