Two natural molecules preferentially inhibit azole-resistant Candida albicans with MDR1 hyperactivation
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摘要
Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B(RAB) and riccardin D(RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.
Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B(RAB) and riccardin D(RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.
Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B(RAB) and riccardin D(RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.
引文
[1]Garber G.An overview of fungal infections[J].Drugs,2001,61(1):1-12.
[2]Pfaller MA,Diekema DJ.Epidemiology of invasive Candidiasis:a persistent public health problem[J].Clin Microbiol Rev,2007,20(1):133-163.
[3]Pfaller MA,Diekema DJ.Epidemiology of invasive mycoses in North America[J].Crit Rev Microbiol,2010,36(1):1-53.
[4]Loeffler J,Stevens DA.Antifungal drug resistance[J].Clin Infect Dis,2003,36(Supplement_1):S31-S41.
[5]Rodriguez L,Bustamante B,Huaroto L,et al.A multi-centric study of Candida bloodstream infection in Lima-Callao,Peru:Species distribution,antifungal resistance and clinical outcomes[J].PLoS ONE,2017,12(4):e175172.
[6]Sanglard D,Odds FC.Resistance of Candida species to antifungal agents:molecular mechanisms and clinical consequences[J].Lancet Infect Dis,2002,2(2):73-85.
[7]Imamovic L,Sommer MO.Use of collateral sensitivity networks to design drug cycling protocols that avoid resistance development[J].Sci Transl Med,2013,5(204):132r-204r.
[8]Munck C,Gumpert HK,Wallin AIN,et al.Prediction of resistance development against drug combinations by collateral responses to component drugs[J].Sci Transl Med,2014,6(262):156r-262r.
[9]Pál C,Papp B,Lázár V.Collateral sensitivity of antibiotic-resistant microbes[J].Trend Microbiol,2015,23(7):401-407.
[10]Stone LK,Baym M,Lieberman TD,et al.Compounds that select against the tetracycline-resistance efflux pump[J].Nat Chem Biol,2016,12(11):902-904.
[11]Bauer A,Br?nstrup M.Industrial natural product chemistry for drug discovery and development[J].Nat Prod Rep,2014,31(1):35-60.
[12]Sun LM,Sun SJ,Cheng AX,et al.In vitro activities of retigeric acid B alone and in combination with azole antifungal agents against Candida albicans[J].Antimicrob Agents Chemother,2009,53(4):1586-1591.
[13]Wu XZ,Cheng AX,Sun LM,et al.Plagiochin E,an antifungal bis(bibenzyl),exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans[J].Biochim Biophys Acta,2009,1790(8):770-777.
[14]Chang WQ,Zhang M,Li Y,et al.Lichen endophyte derived pyridoxatin inactivates Candida growth by interfering with ergosterol biosynthesis[J].Biochim Biophys Acta,2015,1850(9):1762-1771.
[15]Li Y,Chang WQ,Zhang M,et al.Diorcinol D exerts fungicidal action against Candida albicans through cytoplasm membrane destruction and ROS accumulation[J].PLoS ONE,2015,10(6):e128693.
[16]Zhou YH,Zhang M,Zhu RX,et al.Heptaketides from an endolichenic fungus Biatriospora sp.and their antifungal activity[J].J Nat Prod,2016,79(9):2149-2157.
[17]Perea S,López-Ribot JL,Kirkpatrick WR,et al.Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients[J].Antimicrob Agents Chemother,2001,45(10):2676-2684.
[18]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[J].PLoSPathog,2007,3(11):e164.
[19]Wayne PA.Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts:third edition(M27-A3)[M].Wayne,PA:Clinical and Laboratory Standards Institute,2008.
[20]Li SW,Shi HZ,Chang WQ,et al.Eudesmane sesquiterpenes from Chinese liverwort are substrates of Cdrs and display antifungal activity by targeting Erg6 and Erg11 of Candida albicans[J].Bioorgan Med Chem,2017,25(20):5764-5771.
[21]Zhang JZ,Qiao YN,Li L,et al.ent-Eudesmane-type sesquiterpenoids from the Chinese liverwort Chiloscyphus polyanthus var.rivularis[J].Planta Med,2016,82(11/12):1128-1133.
[22]Arthington-Skaggs BA,Jradi HDT,Morrison CJ.Quantitation of ergosterol content:novel method for determination of fluconazole susceptibility of Candida albicans[J].J Clin Microbiol,1999,37(10):3332-3337.
[23]Yan L,Zhang JD,Li MH,et al.DNA microarray analysis of fluconazole resistance in a laboratory Candida albicans strain[J].Acta Biochim Biophys Sin,2008,40(12):1048-1060.
[24]Quartacci MF,Gli?i?O,Stevanovi?B,et al.Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration[J].J Exp Bot,2002,53(378):2159-2166.
[25]Trinel P,Maes E,Zanetta J,et al.Candida albicans phospholipomannan,a new member of the fungal mannose inositol phosphoceramide family[J].J Biol Chem,2002,277(40):37260-37271.
[26]Sanglard D,Ischer F,Monod M,et al.Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors[J].Antimicrob Agents Chemother,1996,40(10):2300-2305.
[27]Calabrese D,Bille J,Sanglard D.A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans(FLU1)conferring resistance to fluconazole[J].Microbiology,2000,146(11):2743-2754.
[28]Holmes E,Wilson ID,Nicholson JK.Metabolic phenotyping in health and disease[J].Cell,2008,134(5):714-717.
[29]Siikala E,Rautemaa R,Richardson M,et al.Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-andidiasis-ectodermal dystrophy(APECED)patients[J].J Antimicrob Chemoth,2010,65(12):2505-2513.
[30]Flowers SA,Barker KS,Berkow EL,et al.Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans[J].Eukaryot Cell,2012,EC-00215-12.
[31]Badolo L,Jensen B,S?ll C,et al.Evaluation of 309 molecules as inducers of CYP3A4,CYP2B6,CYP1A2,OATP1B1,OCT1,MDR1,MRP2,MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture[J].Xenobiotica,2015,45(2):177-187.
[32]Silve A,Mir LM.Cell electropermeabilization and cellular uptake of small molecules:the electrochemotherapy concept[M].In:Kee S,Gehl J,Lee E(eds).Clinical aspects of electroporation.Springer,New York,2011:69-82.
[33]Hitchcock CA,Barrett-Bee KJ,Russell NJ,et al.The Lipid composition of azole-sensitive and azole-resistant strains of Candida albicans[J].Microbiology,1986,132(9):2421-2431.
[34]L?ffler J,Einsele H,Hebart H,et al.Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains[J].FEMS Microbiol Lett,2000,185(1):59-63.
[35]Suutari M,Laakso S.Microbial fatty acids and thermal adaptation[J].Crit Rev Microbiol,1994,20(4):285-328.
[36]Suutari M,Liukkonen K,Laakso S.Temperature adaptation in yeasts:the role of fatty acids[J].Microbiology,1990,136(8):1469-1474.
[37]Sun LM,Lv BB,Cheng AX,et al.The effect of Plagiochin Ealone and in combination with fluconazole on the ergosterol biosynthesis of Candida albicans[J].Biol Pharm Bull,2009,32(1):36-40.
[38]Sun LM,Cheng AX,Wu XZ,et al.Synergistic mechanisms of retigeric acid B and azoles against Candida albicans[J].J Appl Microb,2009,108(1):341-348.
[39]Louw GE,Warren RM,Gry van Pittius NC,et al.A balancing act:efflux/influx in mycobacterial drug resistance[J].Antimicrob Agents Chemother,2009,53(8):3181-3189.
[40]Mukhopadhyay K,Prasad T,Saini P,et al.Membrane sphingolipid-ergosterol interactions are important determinants of multidrug resistance in Candida albicans[J].Antimicrob Agents Chemother,2004,48(5):1778-1787.
[41]Kohli A,Smriti,Mukhopadhyay K,et al.In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry[J].Antimicrob Agents Chemother,2002,46(4):1046-1052.
[42]Chang WQ,Li Y,Zhang L,et al.Retigeric acid B attenuates the virulence of Candida albicans via inhibiting adenylyl cyclase activity targeted by enhanced farnesol production[J].PLoS ONE,2012,7(7):e41624.
[2]Pfaller MA,Diekema DJ.Epidemiology of invasive Candidiasis:a persistent public health problem[J].Clin Microbiol Rev,2007,20(1):133-163.
[3]Pfaller MA,Diekema DJ.Epidemiology of invasive mycoses in North America[J].Crit Rev Microbiol,2010,36(1):1-53.
[4]Loeffler J,Stevens DA.Antifungal drug resistance[J].Clin Infect Dis,2003,36(Supplement_1):S31-S41.
[5]Rodriguez L,Bustamante B,Huaroto L,et al.A multi-centric study of Candida bloodstream infection in Lima-Callao,Peru:Species distribution,antifungal resistance and clinical outcomes[J].PLoS ONE,2017,12(4):e175172.
[6]Sanglard D,Odds FC.Resistance of Candida species to antifungal agents:molecular mechanisms and clinical consequences[J].Lancet Infect Dis,2002,2(2):73-85.
[7]Imamovic L,Sommer MO.Use of collateral sensitivity networks to design drug cycling protocols that avoid resistance development[J].Sci Transl Med,2013,5(204):132r-204r.
[8]Munck C,Gumpert HK,Wallin AIN,et al.Prediction of resistance development against drug combinations by collateral responses to component drugs[J].Sci Transl Med,2014,6(262):156r-262r.
[9]Pál C,Papp B,Lázár V.Collateral sensitivity of antibiotic-resistant microbes[J].Trend Microbiol,2015,23(7):401-407.
[10]Stone LK,Baym M,Lieberman TD,et al.Compounds that select against the tetracycline-resistance efflux pump[J].Nat Chem Biol,2016,12(11):902-904.
[11]Bauer A,Br?nstrup M.Industrial natural product chemistry for drug discovery and development[J].Nat Prod Rep,2014,31(1):35-60.
[12]Sun LM,Sun SJ,Cheng AX,et al.In vitro activities of retigeric acid B alone and in combination with azole antifungal agents against Candida albicans[J].Antimicrob Agents Chemother,2009,53(4):1586-1591.
[13]Wu XZ,Cheng AX,Sun LM,et al.Plagiochin E,an antifungal bis(bibenzyl),exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans[J].Biochim Biophys Acta,2009,1790(8):770-777.
[14]Chang WQ,Zhang M,Li Y,et al.Lichen endophyte derived pyridoxatin inactivates Candida growth by interfering with ergosterol biosynthesis[J].Biochim Biophys Acta,2015,1850(9):1762-1771.
[15]Li Y,Chang WQ,Zhang M,et al.Diorcinol D exerts fungicidal action against Candida albicans through cytoplasm membrane destruction and ROS accumulation[J].PLoS ONE,2015,10(6):e128693.
[16]Zhou YH,Zhang M,Zhu RX,et al.Heptaketides from an endolichenic fungus Biatriospora sp.and their antifungal activity[J].J Nat Prod,2016,79(9):2149-2157.
[17]Perea S,López-Ribot JL,Kirkpatrick WR,et al.Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients[J].Antimicrob Agents Chemother,2001,45(10):2676-2684.
[18]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[J].PLoSPathog,2007,3(11):e164.
[19]Wayne PA.Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts:third edition(M27-A3)[M].Wayne,PA:Clinical and Laboratory Standards Institute,2008.
[20]Li SW,Shi HZ,Chang WQ,et al.Eudesmane sesquiterpenes from Chinese liverwort are substrates of Cdrs and display antifungal activity by targeting Erg6 and Erg11 of Candida albicans[J].Bioorgan Med Chem,2017,25(20):5764-5771.
[21]Zhang JZ,Qiao YN,Li L,et al.ent-Eudesmane-type sesquiterpenoids from the Chinese liverwort Chiloscyphus polyanthus var.rivularis[J].Planta Med,2016,82(11/12):1128-1133.
[22]Arthington-Skaggs BA,Jradi HDT,Morrison CJ.Quantitation of ergosterol content:novel method for determination of fluconazole susceptibility of Candida albicans[J].J Clin Microbiol,1999,37(10):3332-3337.
[23]Yan L,Zhang JD,Li MH,et al.DNA microarray analysis of fluconazole resistance in a laboratory Candida albicans strain[J].Acta Biochim Biophys Sin,2008,40(12):1048-1060.
[24]Quartacci MF,Gli?i?O,Stevanovi?B,et al.Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration[J].J Exp Bot,2002,53(378):2159-2166.
[25]Trinel P,Maes E,Zanetta J,et al.Candida albicans phospholipomannan,a new member of the fungal mannose inositol phosphoceramide family[J].J Biol Chem,2002,277(40):37260-37271.
[26]Sanglard D,Ischer F,Monod M,et al.Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors[J].Antimicrob Agents Chemother,1996,40(10):2300-2305.
[27]Calabrese D,Bille J,Sanglard D.A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans(FLU1)conferring resistance to fluconazole[J].Microbiology,2000,146(11):2743-2754.
[28]Holmes E,Wilson ID,Nicholson JK.Metabolic phenotyping in health and disease[J].Cell,2008,134(5):714-717.
[29]Siikala E,Rautemaa R,Richardson M,et al.Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-andidiasis-ectodermal dystrophy(APECED)patients[J].J Antimicrob Chemoth,2010,65(12):2505-2513.
[30]Flowers SA,Barker KS,Berkow EL,et al.Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans[J].Eukaryot Cell,2012,EC-00215-12.
[31]Badolo L,Jensen B,S?ll C,et al.Evaluation of 309 molecules as inducers of CYP3A4,CYP2B6,CYP1A2,OATP1B1,OCT1,MDR1,MRP2,MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture[J].Xenobiotica,2015,45(2):177-187.
[32]Silve A,Mir LM.Cell electropermeabilization and cellular uptake of small molecules:the electrochemotherapy concept[M].In:Kee S,Gehl J,Lee E(eds).Clinical aspects of electroporation.Springer,New York,2011:69-82.
[33]Hitchcock CA,Barrett-Bee KJ,Russell NJ,et al.The Lipid composition of azole-sensitive and azole-resistant strains of Candida albicans[J].Microbiology,1986,132(9):2421-2431.
[34]L?ffler J,Einsele H,Hebart H,et al.Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains[J].FEMS Microbiol Lett,2000,185(1):59-63.
[35]Suutari M,Laakso S.Microbial fatty acids and thermal adaptation[J].Crit Rev Microbiol,1994,20(4):285-328.
[36]Suutari M,Liukkonen K,Laakso S.Temperature adaptation in yeasts:the role of fatty acids[J].Microbiology,1990,136(8):1469-1474.
[37]Sun LM,Lv BB,Cheng AX,et al.The effect of Plagiochin Ealone and in combination with fluconazole on the ergosterol biosynthesis of Candida albicans[J].Biol Pharm Bull,2009,32(1):36-40.
[38]Sun LM,Cheng AX,Wu XZ,et al.Synergistic mechanisms of retigeric acid B and azoles against Candida albicans[J].J Appl Microb,2009,108(1):341-348.
[39]Louw GE,Warren RM,Gry van Pittius NC,et al.A balancing act:efflux/influx in mycobacterial drug resistance[J].Antimicrob Agents Chemother,2009,53(8):3181-3189.
[40]Mukhopadhyay K,Prasad T,Saini P,et al.Membrane sphingolipid-ergosterol interactions are important determinants of multidrug resistance in Candida albicans[J].Antimicrob Agents Chemother,2004,48(5):1778-1787.
[41]Kohli A,Smriti,Mukhopadhyay K,et al.In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry[J].Antimicrob Agents Chemother,2002,46(4):1046-1052.
[42]Chang WQ,Li Y,Zhang L,et al.Retigeric acid B attenuates the virulence of Candida albicans via inhibiting adenylyl cyclase activity targeted by enhanced farnesol production[J].PLoS ONE,2012,7(7):e41624.