RTA2基因降低白念珠菌对氟康唑敏感性的机制研究
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摘要
在白念珠菌（Candida albicans）中，由于越来越多的菌株对药物产生了耐药性，导致临床上用于治疗念珠菌感染的一线药物氟康唑（Fluconazloe,FLC）的治疗产生失败。我们实验室前期的研究发现：在体外，RTA2基因编码的蛋白参与了由白念珠菌钙调神经磷酸酶（calcineurin,CaN）信号通路介导的对氟康唑的耐药。在本研究中，通过药物敏感性实验考察不同基因背景的RTA2基因相关菌株对氟康唑的敏感性差异，发现：在野生型菌株（cnaΔ/Δ::CNA，crz1Δ/Δ::CRZ1，rta2Δ/Δ::RTA2）中加入1mM CaCl2可以激活CaN信号通路，使菌株对唑类药物氟康唑的敏感性显著降低（最低抑菌浓度由0.5μg/ml升高为16μg/ml和64μg/ml）；加入1mM CaCl2激活CaN信号通路，RTA2基因缺失菌（rta2Δ/Δ,cnaΔ/Δ::CNA；rta2Δ/Δ, crz1Δ/Δ::CRZ1）和钙调神经磷酸酶CNA基因缺失菌（cnaΔ/Δ,rta2Δ/Δ::RTA2）以及CaN信号通路转录因子CRZ1基因缺失菌（crz1Δ/Δ, rta2Δ/Δ::RTA2）一样，对氟康唑的敏感性无显著变化。说明在体外钙离子激活的CaN信号通路通过RTA2p降低白念珠菌对氟康唑的敏感性。通过透射电镜实验考察不同浓度的氟康唑对不同基因背景的RTA2基因相关菌株细胞膜超微结构的影响，我们发现：在体外，钙离子激活钙调神经磷酸酶通路后，可以通过Rta2p来减弱氟康唑对细胞膜的损伤，从而显著的降低了白念珠菌对氟康唑的敏感性。此外，我们构建了小鼠深部真菌感染模型，考察基因RTA2对模型动物生存时间及肾脏荷菌量的影响，发现：RTA2基因本身并不能影响白念珠菌的毒力；但RTA2基因缺失后，感染小鼠接受氟康唑治疗后，生存率显著提高；相反，异位表达RTA2基因显著的降低了氟康唑在宿主中对白念珠菌的疗效。综上所述，我们认为在体内体外钙离子激活的钙调神经磷酸酶通路，通过Rta2p来降低白念珠菌对氟康唑的敏感性。
在白念珠菌中，基因RTA2是钙调神经磷酸酶通路调控的一个下游基因，但是其调控机制并不明确。本课题采用海肾荧光素酶报告基因（Renilla Luciferase，RLUC）系统进行研究：将不同长度的RTA2基因启动子片段连接到报告基因载体中，再将构建好的载体分别转入菌株DJY201（Δ/Δcna,Δ/Δrta2）、MJY201（Δ/Δcrz1,Δ/Δrta2）、JXM101（Δ/Δrta2）中，考察加入钙离子前后荧光活性的变化倍数。进一步证明RTA2基因是CaN信号通路的靶基因，其表达水平受到转录因子Crzlp的调控；发现Crzlp的DNA结合序列—钙调神经磷酸酶依赖性应答元件（calcineruin-dependent responseelement,CDRE），可能位于RTA2基因启动子上游-973bp~-920bp的区间内，通过生物信息学分析推测其序列可能为GATGT。
我们实验室前期研究发现：在临床白念珠菌中，外源性加入1mMCaCl2激活CaN信号通路，可以不同程度的上调RTA2基因的表达水平。通过小鼠深部真菌感染模型发现，钙离子诱导的RTA2基因高表达的菌株感染的小鼠接受氟康唑治疗后，生存率显著低于钙离子诱导的RTA2基因非高表达的菌株感染的小鼠。本研究通过体外药物敏感性实验和实时定量（Real-Time） PCR实验发现：钙离子诱导的菌株对氟康唑敏感性降低和RTA2基因的表达水平密切相关。在钙离子诱导的RTA2基因高表达的临床菌株0511522中，敲除了RTA2基因。通过体外药物敏感性实验发现， RTA2基因缺失菌RTA2N4（△/△rta2）无法产生钙离子介导的对氟康唑的敏感性降低；通过小鼠深部真菌感染模型发现，敲除RTA2基因并不影响菌株的毒力；但是RTA2基因缺失后，感染动物接受氟康唑治疗以后，生存率显著升高。综上所述，在体内体外钙离子通过激活钙调磷酸酶通路诱导RTA2基因高表达从而降低临床菌株对氟康唑的敏感性。
Due to the emergence of drug-resistance, first-line therapy with fluconazole (FLC)increasingly resulted in clinical failure for the treatment of candidemia. Our previousstudies found that in vitro RTA2was involved in the calcineurin-mediated resistance toFLC in C. albicans. In this study, we found that calcium-activated-calcineurin significantlyreduced the in vitro sensitivity of C. albicans to FLC by blocking the impairment of FLCto the plasma membrane via Rta2p. Furthermore, we found that RTA2itself was notinvolved in C. albicans virulence, but the disruption of RTA2dramatically increased thetherapeutic efficacy of FLC in a murine model of systemic candidiasis. Conversely, ectopicexpression of RTA2significantly reduced FLC efficacy in a mammalian host. Finally, wefound that calcium-activated-calcineurin, through its target Rta2p, dramatically reduced theefficacy of FLC against candidemia. Given the critical roles of Rta2p in controlling theefficacy of FLC, Rta2p can be a potential drug target for antifungal therapies.
RTA2was a target of the calcineurin signaling pathway, but its regulatory mechanismwas not clear. We used the Renilla Luciferase reporter system to study this subject:We gotfurther evidence of RTA2was a target gene of CaN signaling pathway and whoseexpression levels was regulated by the transcription factor Crzlp;the CDRE(calcineruin-dependent response element)components of RTA2mainly located in-973bp~-920bp upstream of the promoter of RTA2gene,the sequences might be GATGT.
The previous study found that1mMCaCl2can activate CaN signaling pathway andincreased the expression level of RTA2to different degrees in clinical Candida albicans.Ca2+-induced-upregulation of RTA2dramatically decreased the therapeutic efficacy ofFLC in a murine model of systemic candidiasis compared with Ca2+-induced-nonupregulation of RTA2. In this study, through the in vitro susceptibility testing andReal-Time PCR experiments: we demonstrate that Ca2+-induced-upregulation of RTA2wascorrelated with the in vitro Ca2+-reduced-sensitivity to fluconazole. The expression level ofRTA2was increased by Ca2+in clinical isolate of0511522.The disruption of RTA2in thisisolate block the in vitro Ca2+-reduced-sensitivity to fluconazole; The relationship betweenCa2+-induced-upregulation of RTA2and in vitro Ca2+-reduced-sensitivity to fluconazolewas also verified in vivo. The disruption of RTA2in0511522can significantly increasedthe therapeutic efficacy of FLC in a murine model of systemic candidiasis. To conclude, invitro and in vivo calcium induced the up-regulation of RTA2gene to makes the clinical isolates reduce its sensitivity to fluconazole.
在白念珠菌中，基因RTA2是钙调神经磷酸酶通路调控的一个下游基因，但是其调控机制并不明确。本课题采用海肾荧光素酶报告基因（Renilla Luciferase，RLUC）系统进行研究：将不同长度的RTA2基因启动子片段连接到报告基因载体中，再将构建好的载体分别转入菌株DJY201（Δ/Δcna,Δ/Δrta2）、MJY201（Δ/Δcrz1,Δ/Δrta2）、JXM101（Δ/Δrta2）中，考察加入钙离子前后荧光活性的变化倍数。进一步证明RTA2基因是CaN信号通路的靶基因，其表达水平受到转录因子Crzlp的调控；发现Crzlp的DNA结合序列—钙调神经磷酸酶依赖性应答元件（calcineruin-dependent responseelement,CDRE），可能位于RTA2基因启动子上游-973bp~-920bp的区间内，通过生物信息学分析推测其序列可能为GATGT。
我们实验室前期研究发现：在临床白念珠菌中，外源性加入1mMCaCl2激活CaN信号通路，可以不同程度的上调RTA2基因的表达水平。通过小鼠深部真菌感染模型发现，钙离子诱导的RTA2基因高表达的菌株感染的小鼠接受氟康唑治疗后，生存率显著低于钙离子诱导的RTA2基因非高表达的菌株感染的小鼠。本研究通过体外药物敏感性实验和实时定量（Real-Time） PCR实验发现：钙离子诱导的菌株对氟康唑敏感性降低和RTA2基因的表达水平密切相关。在钙离子诱导的RTA2基因高表达的临床菌株0511522中，敲除了RTA2基因。通过体外药物敏感性实验发现， RTA2基因缺失菌RTA2N4（△/△rta2）无法产生钙离子介导的对氟康唑的敏感性降低；通过小鼠深部真菌感染模型发现，敲除RTA2基因并不影响菌株的毒力；但是RTA2基因缺失后，感染动物接受氟康唑治疗以后，生存率显著升高。综上所述，在体内体外钙离子通过激活钙调磷酸酶通路诱导RTA2基因高表达从而降低临床菌株对氟康唑的敏感性。
Due to the emergence of drug-resistance, first-line therapy with fluconazole (FLC)increasingly resulted in clinical failure for the treatment of candidemia. Our previousstudies found that in vitro RTA2was involved in the calcineurin-mediated resistance toFLC in C. albicans. In this study, we found that calcium-activated-calcineurin significantlyreduced the in vitro sensitivity of C. albicans to FLC by blocking the impairment of FLCto the plasma membrane via Rta2p. Furthermore, we found that RTA2itself was notinvolved in C. albicans virulence, but the disruption of RTA2dramatically increased thetherapeutic efficacy of FLC in a murine model of systemic candidiasis. Conversely, ectopicexpression of RTA2significantly reduced FLC efficacy in a mammalian host. Finally, wefound that calcium-activated-calcineurin, through its target Rta2p, dramatically reduced theefficacy of FLC against candidemia. Given the critical roles of Rta2p in controlling theefficacy of FLC, Rta2p can be a potential drug target for antifungal therapies.
RTA2was a target of the calcineurin signaling pathway, but its regulatory mechanismwas not clear. We used the Renilla Luciferase reporter system to study this subject:We gotfurther evidence of RTA2was a target gene of CaN signaling pathway and whoseexpression levels was regulated by the transcription factor Crzlp;the CDRE(calcineruin-dependent response element)components of RTA2mainly located in-973bp~-920bp upstream of the promoter of RTA2gene,the sequences might be GATGT.
The previous study found that1mMCaCl2can activate CaN signaling pathway andincreased the expression level of RTA2to different degrees in clinical Candida albicans.Ca2+-induced-upregulation of RTA2dramatically decreased the therapeutic efficacy ofFLC in a murine model of systemic candidiasis compared with Ca2+-induced-nonupregulation of RTA2. In this study, through the in vitro susceptibility testing andReal-Time PCR experiments: we demonstrate that Ca2+-induced-upregulation of RTA2wascorrelated with the in vitro Ca2+-reduced-sensitivity to fluconazole. The expression level ofRTA2was increased by Ca2+in clinical isolate of0511522.The disruption of RTA2in thisisolate block the in vitro Ca2+-reduced-sensitivity to fluconazole; The relationship betweenCa2+-induced-upregulation of RTA2and in vitro Ca2+-reduced-sensitivity to fluconazolewas also verified in vivo. The disruption of RTA2in0511522can significantly increasedthe therapeutic efficacy of FLC in a murine model of systemic candidiasis. To conclude, invitro and in vivo calcium induced the up-regulation of RTA2gene to makes the clinical isolates reduce its sensitivity to fluconazole.
引文
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[3]. Brown GD, Dennings DW, Levitz SM.Tackling human fungal infections. Science,2012,336:647.
[4]. Blankenship JR, Steinbach WJ, Perfect JR, Heitman J. Teaching old drugs new tricks: reincarnatingimmunosuppressants as antifungal drugs. Curr Opin Investig Drugs,2003,4:192–199.
[5]. Cowen LE, Singh SD, Kohler JR, Collins C, Zaas AK, et al. Harnessing Hsp90function as apowerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U SA,2009,106:2818–2823.
[6]. Zhai B, Wu C, Wang L, Sachs MS, Lin X. The antidepressant sertraline provides a promisingtherapeutic option for neurotropic cryptococcal infections.Antimicrob Agents Chemother,2012,56:3758–3766.
[7]. Breger J, Fuchs BB, Aperis G, Moy TI, Ausubel FM, et al. Antifungal chemical compoundsidentified using a C. elegans pathogenicity assay. PLoS Pathog,2007,3: e18.doi:10.1371/journal.ppat.0030018
[8]. Spitzer M, Griffiths E, Blakely KM, Wildenhain J, Ejim L, et al. Crossspecies discovery ofsyncretic drug combinations that potentiate the antifungal fluconazole. Mol Syst Biol,2011,7:499.
[9]. Roemer T, Xu D, Singh SB, Parish CA, Harris G, et al. Confronting the challenges of naturalproduct-based antifungal discovery. Chem Biol,2011,18:148–164.
[10]. Tebbets B, Stewart D, Lawry S, Nett J, Nantel A, et al. Identification and characterization ofantifungal compounds using a saccharomyces cerevisiae reporter bioassay. PLoS ONE,2012,7: e36021.doi:10.1371/journal.pone.0036021
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[1]. Lai CC, Tan CK, Huang YT, Shao PL, Hsueh PR. Current challenges in the management ofinvasive fungal infections. J Infect Chemother,2008,14:77–85.
[2]. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, et al. Estimation of the currentglobal burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS,2009,23:525–530.
[3]. Brown GD, Dennings DW, Levitz SM.Tackling human fungal infections. Science,2012,336:647.
[4]. Blankenship JR, Steinbach WJ, Perfect JR, Heitman J. Teaching old drugs new tricks: reincarnatingimmunosuppressants as antifungal drugs. Curr Opin Investig Drugs,2003,4:192–199.
[5]. Cowen LE, Singh SD, Kohler JR, Collins C, Zaas AK, et al. Harnessing Hsp90function as apowerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U SA,2009,106:2818–2823.
[6]. Zhai B, Wu C, Wang L, Sachs MS, Lin X. The antidepressant sertraline provides a promisingtherapeutic option for neurotropic cryptococcal infections.Antimicrob Agents Chemother,2012,56:3758–3766.
[7]. Breger J, Fuchs BB, Aperis G, Moy TI, Ausubel FM, et al. Antifungal chemical compoundsidentified using a C. elegans pathogenicity assay. PLoS Pathog,2007,3: e18.doi:10.1371/journal.ppat.0030018
[8]. Spitzer M, Griffiths E, Blakely KM, Wildenhain J, Ejim L, et al. Crossspecies discovery ofsyncretic drug combinations that potentiate the antifungal fluconazole. Mol Syst Biol,2011,7:499.
[9]. Roemer T, Xu D, Singh SB, Parish CA, Harris G, et al. Confronting the challenges of naturalproduct-based antifungal discovery. Chem Biol,2011,18:148–164.
[10]. Tebbets B, Stewart D, Lawry S, Nett J, Nantel A, et al. Identification and characterization ofantifungal compounds using a saccharomyces cerevisiae reporter bioassay. PLoS ONE,2012,7: e36021.doi:10.1371/journal.pone.0036021
[11]. Krysan DJ, Didone L. A high-throughput screening assay for small molecules that disrupt yeastcell integrity. J Biomol Screen,2008,13:657–664.
[12]. Wiseman H, Cannon M, Arnstein HR.Observation and significance of growth inhibition ofSaccharomyces cerevisiae (A224A) by the anti-oestrogen drug tamoxifen. Biochem Soc Trans,1989,17:1038–1039.
[13]. Dolan K, Montgomery S, Buchheit B, Didone L, Wellington M, et al. Antifungal activity oftamoxifen: in vitro and in vivo activities and mechanistic characterization. Antimicrob AgentsChemother,2009,53:3337–3346.
[14]. Walker SS, Xu Y, Triantafyllou I, Waldman MF, Mendrick C, et al. Discovery of a novel class oforally active antifungal beta-1,3-D-glucan synthase inhibitors. Antimicrob Agents Chemother,2011,55:5099–5106.
[15]. Kitamura A, Someya K, Hata M, Nakajima R, Takemura M.Discovery of a small-moleculeinhibitor of {beta}-1,6-glucan synthesis. Antimicrob Agents Chemother,2009,53:670–677.
[16]. Miyazaki M, Horii T, Hata K, Watanabe NA, Nakamoto K, et al. In vitro activity of E1210, anovel antifungal, against clinically important yeasts and molds. Antimicrob Agents Chemother,2011,55:4652–4658.
[17]. Hata K, Horii T, Miyazaki M, Watanabe NA, Okubo M, et al. Efficacy of oral E1210, a newbroad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis,aspergillosis, and fusariosis. Antimicrob Agents Chemother,2011,55:4543–4551.
[18]. McLellan CA, Whitesell L, King OD, Lancaster AK, Mazitschek R, et al. Inhibiting GPI anchorbiosynthesis in fungi stress the endoplasmic reticulum and enhances immunogencity. ACS ChemBiol,2012, E-pub ahead of print. doi.org/10.1021/cb300235m
[19]. Baxter BK, DiDone L, Ogu D, Schor S, Krysan DJ.Identification, in vitro activity and mode ofaction of phosphoinositide-dependent-1kinase inhibitors as antifungal molecules. ACS Chem Biol,2011,6:502–510.