耐药白念珠菌CYP51酶的活性研究
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摘要
羊毛甾醇14α-去甲基化酶(CYP51) 是真菌细胞膜必需成分-麦角甾
醇生物合成过程中的一个关键酶。氟康唑、伊曲康唑、酮康唑等目前临床最常
用的唑类抗真菌药物都是通过抑制这个靶酶来发挥抗真菌作用的。研究发现
CYP51的结构变异能导致真菌耐药性的产生，因此研究CYP51的结构和功能
关系是指导真菌耐药机制研究和抗真菌药研发的有效途径之一。 ．
研究CYP51变异与白念珠菌耐药的关系，可采取两种策略：① CYP51定点
突变：根据对CYP51活性中心的研究，选取反应活性中心的氨基酸残基，用重
组PCR技术将其突变，考察突变型CYP5l的活性和功能上的变化。② 比较耐药
菌株和敏感菌株CYP5l活性差异：采用敏感菌株、临床耐药菌株和氟康唑诱导
的实验室耐药菌株，比较其CYP51活性和对氟康唑敏感性的差异，而后再研究
其CYP5l的变异情况。
本文在第一部分中利用体外氟康唑诱导获得了耐药白念珠菌，并采用微量
液基稀释药敏实验法，测定了氟康唑对几株白念珠菌的最小抑菌浓度(MIC80) ，
包括敏感菌株和临床耐药株、实验室耐药株，并检测了其对不同药物的交叉耐
药性。敏感白念珠菌在含氟康唑的培养液中传代培养60天就会获得高度而耐药
性，其MIC80可达到≥128μ／ml(敏感菌株MIC值一般<4μg／m1) 。利用该实验
室诱导的耐药白念珠菌，研究其CYP51酶活性，并与亲本敏感株相比较。
第二部分中我们建立了气一质联用测定法(GC-MS)，并用GC-MS法对
敏感和耐药的白念珠菌细胞膜甾醇类物质(CYP51酶的底物和产物)的含量
进行了检测。敏感株在无氟康唑的情况下，细胞膜的麦角甾醇成分含量占总甾
醇类物质的60％以上，而随着氟康唑浓度的增大，CYP51酶受抑制，麦角甾
醇成分的降低和羊毛甾醇等成分的增加与氟康唑呈剂量依赖性关系。在白念珠
菌麦角甾醇生物合成通路中，Trimethyl(24-methylenelanost-8-en-3-o1) 即24(28) -
亚甲基-24，25-二氢羊毛甾醇是CYP51酶的直接反应底物，随氟康唑剂量增加，
其在胞膜中的累积增加量最为明显。而对氟康哗耐药的菌株，细胞膜中踊醇类
的组成与敏感株差异显著，其中y01． 09▲中的麦角甾醇含量仅为30％左右；氟
康唑作用后，细胞膜中的各甾醇成分含量的变化与敏感菌株相比，也有显著的
二 差异，提示真菌细胞膜中涮醇成分的变化与其耐药性的形成有一定的关系。
为进一步定量研究耐药和敏感真菌CYP51酶的活性差异，我们又利用［l－“C」
乙酸整体细胞掺入实验和离细胞酶「2－乙甲羟戊酸掺入实验分别测定了氟康
哗对不同菌株胞膜中麦角吊醇生物合成的半数抑制浓度u0％inhibitLn of
incorporation，IC扣人在真菌整体细胞的卜 口乙酸掺入实验中，随着氟康哗
浓度的提高，敏感菌胞膜中麦角由醇含量减少与羊毛洗醇及其上游成分含量的
累积呈明显的剂量依赖性，氟康哗对 yol．09麦角沿醇生物合成的r。仅为
16．0旭加，说明敏感白念珠菌的CYP51酶对氟康哗的敏感性高。而对于其它
几株耐药的白念珠菌，在相同实验条件下，氟康哗对其作用减弱，其IC扣值有
不同程度的提高，其中对010213＼ IC50达到了 313．3 og／of，与亲本敏感株
相比提高显著。在离细胞酶「2－’川甲羟戊酸掺入实验中，氟康哗对敏感株
y01．09麦角涮醇生物合成的IC50为19．709／01，对实验室耐药株yol．09‘的IC；。
为28．sng／ml，两者有显著差异，对其它耐药株的IC。。增加更为明显，其中对
010213‘的 IC；。则达至了 183．0 ny／＊1。提示 y01．09‘和其它耐药株的 CYP51酶
活性有明显差异。
本课题的目的是建立研究 CYP51对麦角沿醇生物合成活性的可靠方法，为
研究定点突变及耐药菌中CYP51的活性变化奠定基础。上述研究为真菌CYP51
酶的结构和功能研究奠定了基础。在后续的实验中，我们将进一步对耐药菌的
CYPSI基回进行测序比较，为研究真菌耐药机制，寻找广谱、高效、低毒的抗
真菌新药奠定基础。
The cytochrome P450 14ot-lanosterol demethylase (CYP51) of fungi is involved in an important step in the biosynthesis of ergosterol which is essential for fungi to build their plasma membrane and is the target enzyme of azole drugs (fluconazole, ketoconazole and itraconazole, etc.), but it is prone to alterations leading to resistance to these agents. Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the HIV-infected population.We have been interested in understanding the molecular mechanisms of azole resistance in Candida albicans , so it is important to study the structure and function of CYPSl.Two strategies were adopted to research for it: Site-directed mutagenesis was performed to estimate effect of each of those mutations on resistance to azole derivatives.T315A, R467K, G464S mutant CYP51 have been researched by this mothod. (2) To find the mutagenesis from resistant clinical isolates or from induced-resistant strains in vitro.
In this study, several Candida albicans isolates (include azole-resistant and susceptible strains) were tested. We obtained fluconazole-resistant strains(MIC 128ug/ml) induced by serial subcultures in YEPD containing different concentration of fluconazole. Sterol isolation and identification were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the cause for the drug tolerance. When grown in the absence of fluconazole, two sensitive strains and one resistant strain were found to contain ergosterol as their predominant sterol.In contrast, the ergosterol levels in two resistant isolates were comparatively low. When exposure to fluconazole, the decrease in the ergosterol level and the rise in the trimethyl(24-methylenelanost-8-en-3-ol) level were dosage-dependence, but there were many differences between azole-resistant and susceptible strains. It suggested the CYP51 of resistant isolates was less sensitive to fluconazole.
The inhibitory effect of fluconazole on ergosterol biosynthesis in whole-cell fungi was measured by the incorporation of [l-14C]acetate into nonsaponifiable
5
lipids(NSLs). Compared to sensitive isolates, the half inhibitory concentration(IC5o)of fluconazole for ergosterol rise obviously in resistant isolates. IC5o to 010213A was 313.3 ng/ml. While the effect in cell-free extracts was measured by the incorporation of [2-'4C]mevalonate into NSLs. Compared to azole-susceptible strains, ICjo to CYP51 rised significant in azole-resistant strains. y01.09A from 28.5 ng/ml to 40.0 ng/ml, but others rised more significant .There were differences between azole- resistant strains. It suggested that the CYP51 of y01 .09A distinct from other azole-resistant strains.
The aim of this topic is to establish a reliable method for the research of CYP51 on biosynthesis of ergosterol, settle foundation for the research of mutagenesis in resistant fungi and the structure and function of CYP51. In successive experiments, we will sequence and compare the CYP51 genes of the resistant isolates to inspect the molecular mechanisms of azole resistance in Candida albicans.
醇生物合成过程中的一个关键酶。氟康唑、伊曲康唑、酮康唑等目前临床最常
用的唑类抗真菌药物都是通过抑制这个靶酶来发挥抗真菌作用的。研究发现
CYP51的结构变异能导致真菌耐药性的产生，因此研究CYP51的结构和功能
关系是指导真菌耐药机制研究和抗真菌药研发的有效途径之一。 ．
研究CYP51变异与白念珠菌耐药的关系，可采取两种策略：① CYP51定点
突变：根据对CYP51活性中心的研究，选取反应活性中心的氨基酸残基，用重
组PCR技术将其突变，考察突变型CYP5l的活性和功能上的变化。② 比较耐药
菌株和敏感菌株CYP5l活性差异：采用敏感菌株、临床耐药菌株和氟康唑诱导
的实验室耐药菌株，比较其CYP51活性和对氟康唑敏感性的差异，而后再研究
其CYP5l的变异情况。
本文在第一部分中利用体外氟康唑诱导获得了耐药白念珠菌，并采用微量
液基稀释药敏实验法，测定了氟康唑对几株白念珠菌的最小抑菌浓度(MIC80) ，
包括敏感菌株和临床耐药株、实验室耐药株，并检测了其对不同药物的交叉耐
药性。敏感白念珠菌在含氟康唑的培养液中传代培养60天就会获得高度而耐药
性，其MIC80可达到≥128μ／ml(敏感菌株MIC值一般<4μg／m1) 。利用该实验
室诱导的耐药白念珠菌，研究其CYP51酶活性，并与亲本敏感株相比较。
第二部分中我们建立了气一质联用测定法(GC-MS)，并用GC-MS法对
敏感和耐药的白念珠菌细胞膜甾醇类物质(CYP51酶的底物和产物)的含量
进行了检测。敏感株在无氟康唑的情况下，细胞膜的麦角甾醇成分含量占总甾
醇类物质的60％以上，而随着氟康唑浓度的增大，CYP51酶受抑制，麦角甾
醇成分的降低和羊毛甾醇等成分的增加与氟康唑呈剂量依赖性关系。在白念珠
菌麦角甾醇生物合成通路中，Trimethyl(24-methylenelanost-8-en-3-o1) 即24(28) -
亚甲基-24，25-二氢羊毛甾醇是CYP51酶的直接反应底物，随氟康唑剂量增加，
其在胞膜中的累积增加量最为明显。而对氟康哗耐药的菌株，细胞膜中踊醇类
的组成与敏感株差异显著，其中y01． 09▲中的麦角甾醇含量仅为30％左右；氟
康唑作用后，细胞膜中的各甾醇成分含量的变化与敏感菌株相比，也有显著的
二 差异，提示真菌细胞膜中涮醇成分的变化与其耐药性的形成有一定的关系。
为进一步定量研究耐药和敏感真菌CYP51酶的活性差异，我们又利用［l－“C」
乙酸整体细胞掺入实验和离细胞酶「2－乙甲羟戊酸掺入实验分别测定了氟康
哗对不同菌株胞膜中麦角吊醇生物合成的半数抑制浓度u0％inhibitLn of
incorporation，IC扣人在真菌整体细胞的卜 口乙酸掺入实验中，随着氟康哗
浓度的提高，敏感菌胞膜中麦角由醇含量减少与羊毛洗醇及其上游成分含量的
累积呈明显的剂量依赖性，氟康哗对 yol．09麦角沿醇生物合成的r。仅为
16．0旭加，说明敏感白念珠菌的CYP51酶对氟康哗的敏感性高。而对于其它
几株耐药的白念珠菌，在相同实验条件下，氟康哗对其作用减弱，其IC扣值有
不同程度的提高，其中对010213＼ IC50达到了 313．3 og／of，与亲本敏感株
相比提高显著。在离细胞酶「2－’川甲羟戊酸掺入实验中，氟康哗对敏感株
y01．09麦角涮醇生物合成的IC50为19．709／01，对实验室耐药株yol．09‘的IC；。
为28．sng／ml，两者有显著差异，对其它耐药株的IC。。增加更为明显，其中对
010213‘的 IC；。则达至了 183．0 ny／＊1。提示 y01．09‘和其它耐药株的 CYP51酶
活性有明显差异。
本课题的目的是建立研究 CYP51对麦角沿醇生物合成活性的可靠方法，为
研究定点突变及耐药菌中CYP51的活性变化奠定基础。上述研究为真菌CYP51
酶的结构和功能研究奠定了基础。在后续的实验中，我们将进一步对耐药菌的
CYPSI基回进行测序比较，为研究真菌耐药机制，寻找广谱、高效、低毒的抗
真菌新药奠定基础。
The cytochrome P450 14ot-lanosterol demethylase (CYP51) of fungi is involved in an important step in the biosynthesis of ergosterol which is essential for fungi to build their plasma membrane and is the target enzyme of azole drugs (fluconazole, ketoconazole and itraconazole, etc.), but it is prone to alterations leading to resistance to these agents. Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the HIV-infected population.We have been interested in understanding the molecular mechanisms of azole resistance in Candida albicans , so it is important to study the structure and function of CYPSl.Two strategies were adopted to research for it: Site-directed mutagenesis was performed to estimate effect of each of those mutations on resistance to azole derivatives.T315A, R467K, G464S mutant CYP51 have been researched by this mothod. (2) To find the mutagenesis from resistant clinical isolates or from induced-resistant strains in vitro.
In this study, several Candida albicans isolates (include azole-resistant and susceptible strains) were tested. We obtained fluconazole-resistant strains(MIC 128ug/ml) induced by serial subcultures in YEPD containing different concentration of fluconazole. Sterol isolation and identification were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the cause for the drug tolerance. When grown in the absence of fluconazole, two sensitive strains and one resistant strain were found to contain ergosterol as their predominant sterol.In contrast, the ergosterol levels in two resistant isolates were comparatively low. When exposure to fluconazole, the decrease in the ergosterol level and the rise in the trimethyl(24-methylenelanost-8-en-3-ol) level were dosage-dependence, but there were many differences between azole-resistant and susceptible strains. It suggested the CYP51 of resistant isolates was less sensitive to fluconazole.
The inhibitory effect of fluconazole on ergosterol biosynthesis in whole-cell fungi was measured by the incorporation of [l-14C]acetate into nonsaponifiable
5
lipids(NSLs). Compared to sensitive isolates, the half inhibitory concentration(IC5o)of fluconazole for ergosterol rise obviously in resistant isolates. IC5o to 010213A was 313.3 ng/ml. While the effect in cell-free extracts was measured by the incorporation of [2-'4C]mevalonate into NSLs. Compared to azole-susceptible strains, ICjo to CYP51 rised significant in azole-resistant strains. y01.09A from 28.5 ng/ml to 40.0 ng/ml, but others rised more significant .There were differences between azole- resistant strains. It suggested that the CYP51 of y01 .09A distinct from other azole-resistant strains.
The aim of this topic is to establish a reliable method for the research of CYP51 on biosynthesis of ergosterol, settle foundation for the research of mutagenesis in resistant fungi and the structure and function of CYP51. In successive experiments, we will sequence and compare the CYP51 genes of the resistant isolates to inspect the molecular mechanisms of azole resistance in Candida albicans.
引文
1． Stevens DA. Therapy for opportunistic fungal infectious: past, present and future. Indian J Cancer, 1995． 32(1) : 1-9
2． Powderly,W.G.Keath,M.Sokol Anderson et al.AmphotericinB-resistant Cyptococcus neoformans in a patient with AIDS.Infect.Dis.Clin.Pract. 1990,1:314-316
3． Louie A, Kaw P, Banerjee P. Liu W, Chen G, Miller MH. Impact of the order of initiation of fluconazole and amphotericin B in sequential or combination therapy on killing of Candida albicans in vitro and in a rabbit model of endocarditis and pyelonephritis. Antimicrob Agents Chemother 2001,45(2) : 485-94
4． Yamaguchi H. Molecular and biochemical mechanisms of drug resistance in fungi. Nippon Ishinkin Gakkai Zasshi 1999,40(4) : 199-208
5． Perea S,Lopez-Robit 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 HIV-infected patients. Antimicrob Agents Chemother 2001 ,45(10) :2676-84
6． Asai K, Tsuchimori N, Okonogi K, Perfect JR, Gotoh 0, Yoshida Y. Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450． Antimicrob Agents Chemother 1999, 43(5) : 1163-1169
7． Edlind TD, Henry KW, Metera KA, Katiyar SK. Aspergillus fumigatus CYP51 sequence: potential basis for fluconazole resistance. Med Mycol 2001, 39(3) : 299-302
8． Dominique Sanglard, Francoise Ischer, Luc Koymans et al.Amino acid substitutions in the cytochrome P-450 lanosterol 14 a-demethylase(CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents.Antimicrobial Agents and Chemotherapy, 1998． 42(2) :241-253
9． Patrick Marichal et al. Contribution of mutations in the cytochrome P450 14α-demethylase(Ergl lp,Cyp51p)to azole resistance in Candida albicans.Microbiology, 1999,145:2701-2713
10． David C.Lamb,Diane E.Kelly et al.The mutation T315 in Candida albicans sterol 14α-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.J.Biol.Chem.1997, 272:5682-5688
11． 王文莉，李若瑜，王端礼等，念珠菌对唑类药物耐药机理的探讨[J]．中华皮 肤科杂志，1997． 30(5) ：306
12． Maebashi K, Niimi M, Kudoh M et al. Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients. J Antimicrob Chemother 2001, 47(5) : 527-36
13． Marr KA, Lyons CN. Ha K, Rustad TR, White TC. Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans. Antimicrob Agents Chemother 2001,45(1) : 52-9
14． Paterson PJ, McWhinney PH, Potter M, Kibbler CC, Prentice HG. The combination of oral amphotericin B with azoles prevents the emergence of resistant Candida species in neutropenic patients. Br J Haematol 2001, 112(1) : 175-80
15． Kinoshita H et al.20th International congress of chemotherapy. 1st ed,Australia:International Academic Publishers, 1997:48
16． Granier F. Invasive fungal infections. Epidemiology and new therapies. Presse Med 2000． 29(37) : 2051-2056
17． D'Auria FD, Tecca M, Strippoli R, Simonetti N. In vitro activity of propyl gallate-azole drug combination against fluconazole-and itraconazole-resistant Candida albicans strains. Lett Appl Microbiol 2001, 32(4) : 220-3
18． Kohli A, Smriti, Mukhopadhyay K, Rattan A. et al. In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrob Agents Chemother 2002 Apr; 46(4) : 1046
19． Loffler J, Einsele H, Hebart H,et al. Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains. FEMS Microbiol Lett 2000 Apr 1; 185(1) : 59
20． Maebashi K, Niimi M, Kudoh M,et al. Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients. J Antimicrob Chemother2001 May; 47(5) : 527
2． Powderly,W.G.Keath,M.Sokol Anderson et al.AmphotericinB-resistant Cyptococcus neoformans in a patient with AIDS.Infect.Dis.Clin.Pract. 1990,1:314-316
3． Louie A, Kaw P, Banerjee P. Liu W, Chen G, Miller MH. Impact of the order of initiation of fluconazole and amphotericin B in sequential or combination therapy on killing of Candida albicans in vitro and in a rabbit model of endocarditis and pyelonephritis. Antimicrob Agents Chemother 2001,45(2) : 485-94
4． Yamaguchi H. Molecular and biochemical mechanisms of drug resistance in fungi. Nippon Ishinkin Gakkai Zasshi 1999,40(4) : 199-208
5． Perea S,Lopez-Robit 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 HIV-infected patients. Antimicrob Agents Chemother 2001 ,45(10) :2676-84
6． Asai K, Tsuchimori N, Okonogi K, Perfect JR, Gotoh 0, Yoshida Y. Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450． Antimicrob Agents Chemother 1999, 43(5) : 1163-1169
7． Edlind TD, Henry KW, Metera KA, Katiyar SK. Aspergillus fumigatus CYP51 sequence: potential basis for fluconazole resistance. Med Mycol 2001, 39(3) : 299-302
8． Dominique Sanglard, Francoise Ischer, Luc Koymans et al.Amino acid substitutions in the cytochrome P-450 lanosterol 14 a-demethylase(CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents.Antimicrobial Agents and Chemotherapy, 1998． 42(2) :241-253
9． Patrick Marichal et al. Contribution of mutations in the cytochrome P450 14α-demethylase(Ergl lp,Cyp51p)to azole resistance in Candida albicans.Microbiology, 1999,145:2701-2713
10． David C.Lamb,Diane E.Kelly et al.The mutation T315 in Candida albicans sterol 14α-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.J.Biol.Chem.1997, 272:5682-5688
11． 王文莉，李若瑜，王端礼等，念珠菌对唑类药物耐药机理的探讨[J]．中华皮 肤科杂志，1997． 30(5) ：306
12． Maebashi K, Niimi M, Kudoh M et al. Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients. J Antimicrob Chemother 2001, 47(5) : 527-36
13． Marr KA, Lyons CN. Ha K, Rustad TR, White TC. Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans. Antimicrob Agents Chemother 2001,45(1) : 52-9
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