Cis-2-dodecenoic Acid Mediates Its Synergistic Effect with Triazoles by Interfering with Efflux Pumps in Fluconazole-resistant Candida albicans
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摘要
Objective To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid(BDSF) and fluconazole(FLU) or itraconazole(ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo. Methods Minimum inhibitory concentrations(MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction(qR T-PCR). Results BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ≤ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qR T-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mR NA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis. Conclusion The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.
Objective To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid(BDSF) and fluconazole(FLU) or itraconazole(ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo. Methods Minimum inhibitory concentrations(MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction(qR T-PCR). Results BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ≤ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qR T-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mR NA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis. Conclusion The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.
Objective To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid(BDSF) and fluconazole(FLU) or itraconazole(ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo. Methods Minimum inhibitory concentrations(MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction(qR T-PCR). Results BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ≤ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qR T-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mR NA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis. Conclusion The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.
引文
1.Nucci M,Queiroztelles F,Alvaradomatute T,et al.Epidemiology of Candidemia in Latin America:ALaboratory-Based Survey.PLo S One,2013;8,e59373.
2.Pfaller M,Diekema D.Epidemiology of invasive candidiasis:a persistent public health problem.Clin Microbiol Rev,2007;20,133-63.
3.Pfaller MA,Moet GJ,Messer SA,et al.Geographic variations in species distribution and echinocandin and azole antifungal resistance rates among Candida bloodstream infection isolates:report from the sentry antimicrobial surveillance program(2008 to 2009).J Clin Microbiol,2011;49,396-9.
4.Matsubara VH,Wang Y,Bandara H,et al.Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth,cell adhesion,and filamentation.Appl Microbiol Biotechnol,2016;100,6415-26.
5.López-Martínez R.Candidosis,a new challenge.Clin Dermatol,2010;28,178-84.
6.Akins RA,Sobel JD.Antifungal Targets,Mechanisms of Action,and Resistance in Candida albicans.Antimicrobial Drug Resistance,2017;429-75.
7.Del Sorbo G,Schoonbeek HJ,De Waard MA.Fungal transporters involved in efflux of natural toxic compounds and fungicides.Fungal Genet Biol,2000;30,1-15.
8.Lohberger A,Coste AT,Sanglard D.Distinct roles of Candida albicans drug resistance transcription factors TAC1,MRR1,and UPC2 in virulence.Eukaryot Cell,2014;13,127-42.
9.Dib L,Hayek P,Sadek H,et al.The Candida albicans Ddr48protein is essential for filamentation,stress response,and confers partial antifungal drug resistance.Med Sci Monit,2008;14,Br113-21.
10.Heimark L,Shipkova P,Greene J,et al.Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis.J Mass Spectrom,2002;37,265-9.
11.Hoot SJ,Oliver BG,White TC.Candida albicans UPC2 is transcriptionally induced in response to antifungal drugs and anaerobicity through Upc2p-dependent and-independent mechanisms.Microbiology,2008;154,2748-56.
12.Znaidi S,Weber S,Al-Abdin OZ,et al.Genomewide location analysis of Candida albicans Upc2p,a regulator of sterol metabolism and azole drug resistance.Eukaryot Cell,2008;7,836-47.
13.Denning D.Can we prevent azole resistance in fungi?The Lancet,1995;346,454-5.
14.Ejim L,Farha MA,Falconer SB,et al.Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy.Nat Chem Biol,2011;7,348.
15.Butts A,Krysan DJ.Antifungal drug discovery:something old and something new.PLo S Pathog,2012;8,e1002870.
16.Li DD,Xu Y,Zhang DZ,et al.Fluconazole assists berberine to kill fluconazole-resistant Candida albicans.Antimicrob Agents Chemother,2013;57,6016-27.
17.Mukherjee PK,Sheehan DJ,Hitchcock CA,et al.Combination treatment of invasive fungal infections.Clin Microbiol Rev,2005;18,163-94.
18.Kabir MA,Ahmad Z.Candida infections and their prevention.ISRN preventive medicine,2012;763628.
19.Azevedo MM,Teixeira-Santos R,Silva AP,et al.The effect of antibacterial and non-antibacterial compounds alone or associated with antifugals upon fungi.Front Microbiol,2015;6,669.
20.Guo Q,Sun S,Yu J,et al.Synergistic activity of azoles with amiodarone against clinically resistant Candida albicans tested by chequerboard and time-kill methods.J Med Microbiol,2008;57,457-62.
21.Gao Y,Li H,Liu S,et al.Synergistic effect of fluconazole and doxycycline against Candida albicans biofilms resulting from calcium fluctuation and downregulation of fluconazole-inducible efflux pump gene overexpression.J Med Microbiol,2014;63,956-61.
22.Li Y,Wan Z,Liu W,et al.Synergistic activity of chloroquine with fluconazole against fluconazole-resistant isolates of Candida species.Antimicrob Agents Chemother,2015;59,1365-9.
23.Liu S,Hou Y,Chen X,et al.Combination of fluconazole with non-antifungal agents:a promising approach to cope with resistant Candida albicans infections and insight into new antifungal agent discovery.Int J Antimicrob Agents,2014;43,395-402.
24.Zhao F,Dong HH,Wang YH,et al.Synthesis and synergistic antifungal effects of monoketone derivatives of curcumin against fluconazole-resistant Candida spp.Medchemcomm,2017;8,1093-102.
25.P Tegos G,Haynes M,Jacob Strouse J,et al.Microbial efflux pump inhibition:tactics and strategies.Curr Pharm Des,2011;17,1291-302.
26.Nagayoshi Y,Miyazaki T,Shimamura S,et al.Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species.PLo S One,2017;12,e0180990.
27.Xia J,Fang Q,Xu W,et al.In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains.Biofouling,2017;33,283-93.
28.Willers C,Wentzel JF,Du PL,et al.Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms:the role of efflux inhibitors.Expert Opin Ther Targets,2017;21,23.
29.Boon C,Deng Y,Wang LH,et al.A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition.ISME J,2008;2,27.
30.Wang LH,Zhang LH.Inhibitors of yeast filamentous growth and method of their manufacture.United States Patent No.US7915313B2,2014.
31.Zhang Y,Cai C,Yang Y,et al.Blocking of Candida albicans biofilm formation by cis-2-dodecenoic acid and trans-2-dodecenoic acid.J Med Microbiol,2011;60,1643-50.
32.Xu ZH,Liao Y,Li M,et al.Inhibitory effect of BDSF on hyphal growth of clinical Candida albicans.J Microbes Infect,2015;10,247-51.
33.M27-A3 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts;Approved Standard,Third Edition.Clinical and Laboratory Standards Institute.In Wayne,PA,USA;2008.
34.Chen SC,O'donnell ML,Gordon S,et al.Antifungal susceptibility testing using the E test:comparison with the broth macrodilution technique.J Antimicrob Chemoth,1996;37,265-73.
35.Jin J,Guo N,Zhang J,et al.The synergy of honokiol and fluconazole against clinical isolates of azole-resistant Candida albicans.Lett Appl Microbiol,2010;51,351-7.
36.Yang D,Zhang Y,Hu Y,et al.Protective effects of cis-2-dodecenoic acid in an experimental mouse model of vaginal candidiasis.Biomed Environ Sci,2018;31,816-28.
37.Lee SH,Jeon JE,Ahn CH,et al.Inhibition of yeast-to-hypha transition in Candida albicans by phorbasin H isolated from Phorbas sp.Appl Microbiol Biotechnol,2013;97,3141-8.
38.Cannon RD,Lamping E,Holmes AR,et al.Efflux-mediated antifungal drug resistance.Clin Microbiol Rev,2009;22,291-321.
39.Coste AT,Karababa M,Ischer F,et al.TAC1,transcriptional activator of CDR genes,is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2.Eukaryot Cell,2004;3,1639-52.
40.Morschh?user J,Barker KS,Liu TT,et al.The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans.PLo SPathog,2007;3,e164.
41.Sharma M,Prasad R.The quorum-sensing molecule farnesol is a modulator of drug efflux mediated by ABC multidrug transporters and synergizes with drugs in Candida albicans.Antimicrob Agents Ch,2011;55,4834-43.
42.Ahmad A,Khan A,Manzoor N.Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole.Eur J Phar Sc,2013;48,80-6.
43.Simopoulos AP.Essential fatty acids in health and chronic disease.Am J Clin Nutr,1999;70,560S-9S.
2.Pfaller M,Diekema D.Epidemiology of invasive candidiasis:a persistent public health problem.Clin Microbiol Rev,2007;20,133-63.
3.Pfaller MA,Moet GJ,Messer SA,et al.Geographic variations in species distribution and echinocandin and azole antifungal resistance rates among Candida bloodstream infection isolates:report from the sentry antimicrobial surveillance program(2008 to 2009).J Clin Microbiol,2011;49,396-9.
4.Matsubara VH,Wang Y,Bandara H,et al.Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth,cell adhesion,and filamentation.Appl Microbiol Biotechnol,2016;100,6415-26.
5.López-Martínez R.Candidosis,a new challenge.Clin Dermatol,2010;28,178-84.
6.Akins RA,Sobel JD.Antifungal Targets,Mechanisms of Action,and Resistance in Candida albicans.Antimicrobial Drug Resistance,2017;429-75.
7.Del Sorbo G,Schoonbeek HJ,De Waard MA.Fungal transporters involved in efflux of natural toxic compounds and fungicides.Fungal Genet Biol,2000;30,1-15.
8.Lohberger A,Coste AT,Sanglard D.Distinct roles of Candida albicans drug resistance transcription factors TAC1,MRR1,and UPC2 in virulence.Eukaryot Cell,2014;13,127-42.
9.Dib L,Hayek P,Sadek H,et al.The Candida albicans Ddr48protein is essential for filamentation,stress response,and confers partial antifungal drug resistance.Med Sci Monit,2008;14,Br113-21.
10.Heimark L,Shipkova P,Greene J,et al.Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis.J Mass Spectrom,2002;37,265-9.
11.Hoot SJ,Oliver BG,White TC.Candida albicans UPC2 is transcriptionally induced in response to antifungal drugs and anaerobicity through Upc2p-dependent and-independent mechanisms.Microbiology,2008;154,2748-56.
12.Znaidi S,Weber S,Al-Abdin OZ,et al.Genomewide location analysis of Candida albicans Upc2p,a regulator of sterol metabolism and azole drug resistance.Eukaryot Cell,2008;7,836-47.
13.Denning D.Can we prevent azole resistance in fungi?The Lancet,1995;346,454-5.
14.Ejim L,Farha MA,Falconer SB,et al.Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy.Nat Chem Biol,2011;7,348.
15.Butts A,Krysan DJ.Antifungal drug discovery:something old and something new.PLo S Pathog,2012;8,e1002870.
16.Li DD,Xu Y,Zhang DZ,et al.Fluconazole assists berberine to kill fluconazole-resistant Candida albicans.Antimicrob Agents Chemother,2013;57,6016-27.
17.Mukherjee PK,Sheehan DJ,Hitchcock CA,et al.Combination treatment of invasive fungal infections.Clin Microbiol Rev,2005;18,163-94.
18.Kabir MA,Ahmad Z.Candida infections and their prevention.ISRN preventive medicine,2012;763628.
19.Azevedo MM,Teixeira-Santos R,Silva AP,et al.The effect of antibacterial and non-antibacterial compounds alone or associated with antifugals upon fungi.Front Microbiol,2015;6,669.
20.Guo Q,Sun S,Yu J,et al.Synergistic activity of azoles with amiodarone against clinically resistant Candida albicans tested by chequerboard and time-kill methods.J Med Microbiol,2008;57,457-62.
21.Gao Y,Li H,Liu S,et al.Synergistic effect of fluconazole and doxycycline against Candida albicans biofilms resulting from calcium fluctuation and downregulation of fluconazole-inducible efflux pump gene overexpression.J Med Microbiol,2014;63,956-61.
22.Li Y,Wan Z,Liu W,et al.Synergistic activity of chloroquine with fluconazole against fluconazole-resistant isolates of Candida species.Antimicrob Agents Chemother,2015;59,1365-9.
23.Liu S,Hou Y,Chen X,et al.Combination of fluconazole with non-antifungal agents:a promising approach to cope with resistant Candida albicans infections and insight into new antifungal agent discovery.Int J Antimicrob Agents,2014;43,395-402.
24.Zhao F,Dong HH,Wang YH,et al.Synthesis and synergistic antifungal effects of monoketone derivatives of curcumin against fluconazole-resistant Candida spp.Medchemcomm,2017;8,1093-102.
25.P Tegos G,Haynes M,Jacob Strouse J,et al.Microbial efflux pump inhibition:tactics and strategies.Curr Pharm Des,2011;17,1291-302.
26.Nagayoshi Y,Miyazaki T,Shimamura S,et al.Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species.PLo S One,2017;12,e0180990.
27.Xia J,Fang Q,Xu W,et al.In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains.Biofouling,2017;33,283-93.
28.Willers C,Wentzel JF,Du PL,et al.Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms:the role of efflux inhibitors.Expert Opin Ther Targets,2017;21,23.
29.Boon C,Deng Y,Wang LH,et al.A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition.ISME J,2008;2,27.
30.Wang LH,Zhang LH.Inhibitors of yeast filamentous growth and method of their manufacture.United States Patent No.US7915313B2,2014.
31.Zhang Y,Cai C,Yang Y,et al.Blocking of Candida albicans biofilm formation by cis-2-dodecenoic acid and trans-2-dodecenoic acid.J Med Microbiol,2011;60,1643-50.
32.Xu ZH,Liao Y,Li M,et al.Inhibitory effect of BDSF on hyphal growth of clinical Candida albicans.J Microbes Infect,2015;10,247-51.
33.M27-A3 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts;Approved Standard,Third Edition.Clinical and Laboratory Standards Institute.In Wayne,PA,USA;2008.
34.Chen SC,O'donnell ML,Gordon S,et al.Antifungal susceptibility testing using the E test:comparison with the broth macrodilution technique.J Antimicrob Chemoth,1996;37,265-73.
35.Jin J,Guo N,Zhang J,et al.The synergy of honokiol and fluconazole against clinical isolates of azole-resistant Candida albicans.Lett Appl Microbiol,2010;51,351-7.
36.Yang D,Zhang Y,Hu Y,et al.Protective effects of cis-2-dodecenoic acid in an experimental mouse model of vaginal candidiasis.Biomed Environ Sci,2018;31,816-28.
37.Lee SH,Jeon JE,Ahn CH,et al.Inhibition of yeast-to-hypha transition in Candida albicans by phorbasin H isolated from Phorbas sp.Appl Microbiol Biotechnol,2013;97,3141-8.
38.Cannon RD,Lamping E,Holmes AR,et al.Efflux-mediated antifungal drug resistance.Clin Microbiol Rev,2009;22,291-321.
39.Coste AT,Karababa M,Ischer F,et al.TAC1,transcriptional activator of CDR genes,is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2.Eukaryot Cell,2004;3,1639-52.
40.Morschh?user J,Barker KS,Liu TT,et al.The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans.PLo SPathog,2007;3,e164.
41.Sharma M,Prasad R.The quorum-sensing molecule farnesol is a modulator of drug efflux mediated by ABC multidrug transporters and synergizes with drugs in Candida albicans.Antimicrob Agents Ch,2011;55,4834-43.
42.Ahmad A,Khan A,Manzoor N.Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole.Eur J Phar Sc,2013;48,80-6.
43.Simopoulos AP.Essential fatty acids in health and chronic disease.Am J Clin Nutr,1999;70,560S-9S.