ANTIFUNGAL RESPONSE--MDR INDUCTION

真菌可能会以特定的方式对抗真菌化疗的压力做出反应，最终降低治疗的有效性。这种“抗真菌反应”类似于热休克反应，但在机理上不同于热休克反应。这项建议的重点是这样一种抗真菌反应，称为MDR诱导，涉及药物依赖的快速转录诱导编码与多药耐药(MDR)相关的转运体的基因。具体来说，白色念珠菌、克鲁斯假丝酵母菌或酿酒酵母暴露于某些药物(包括抗真菌药物咪康唑和克霉唑、苯并咪唑、阿苯达唑和磺胺嘧啶)15-20分钟后，编码特定MDR转运体的mRNAs增加了3到10倍。初步研究表明，多药耐药诱导是念珠菌对苯并咪唑类药物的内在耐药性以及诱导多药耐药的化合物(阿苯达唑和磺胺嘧啶)与多药耐药转运体底物(氮唑)之间的拮抗作用的最有可能的原因。假说，MDR诱导也可能有助于对唑类药物的表型适应(反映在“拖尾效应”中)，不同真菌对不同氮唑类药物的不同敏感性，以及获得的抗真菌耐药性。这项建议的具体目的是研究：(1)多药耐药诱导的频谱。将测试免疫低下患者常用的新型抗真菌药物和抗感染药物对白色念珠菌的多药耐药诱导和唑类活性的拮抗作用。多药耐药的诱导和拮抗作用将在对唑敏感和耐药的菌株以及白色念珠菌的酵母和菌丝形态中进行比较。从光滑假丝酵母菌、烟曲霉和新生隐球菌中扩增出mdr基因片段，并作为探针用于这些真菌的mdr诱导研究。(2)诱导白念珠菌多药耐药的转录激活子。对酿酒酵母的初步研究表明，有两类转录激活剂参与了唑依赖多药耐药的诱导，即PDR1/PDR3和YAP1/YAP2。这些因子的念珠菌同源物将通过聚合酶链式反应或在酿酒酵母中表达来鉴定。它们在多药耐药诱导中的作用将通过基因破坏来检验。

Fungi may respond to the stress of antifungal chemotherapy in specific ways that ultimately reduce the efficacy of the therapy. This "antifungal response" would be analogous to, but mechanistically distinct from, the heat shock response. The focus of this proposal is one such antifungal response, termed MDR induction, involving the rapid drug-dependent transcriptional induction of genes encoding transporters involved in multidrug resistance (MDR). Specifically, a 15-20 min exposure of Candida albicans, Candida krusei, or Saccharomyces cerevisiae to certain drugs (including the antifungal azoles miconazole and clotrimazole, the benzimidazole albendazole, and sulfadiazine) induced a 3 to > 10-fold increase in mRNAs encoding specific MDR transporters. initial studies indicate that MDR induction is the most likely explanation for the intrinsic resistance of Candida species to benzimidazoles and for the antagonistic activity between compounds that induce MDR (albendazole and sulfadiazine) and compounds that are substrates for the MDR transporters (azoles). Hypothetically, MDR induction could also contribute to phenotypic adaptation to azoles (reflected in the "trailing effect"), to the variable sensitivity of different fungi to different azoles, and to acquired antifungal drug resistance. The Specific Aims of this proposal are to examine the: (1) Spectrum of MDR induction. New antifungal agents and anti-infective agents commonly used in immunocompromised patients will be tested for C. albicans MDR induction and antagonism of azole activity. MDR induction and antagonism will be compared in azole-sensitive and resistant isolates, and in C. albicans yeast and hyphal forms. MDR gene fragments will be amplified from Candida glabrata, Aspergillus fumigatus, and Cryptococcus neoformans and used as probes for MDR induction studies in those fungi. (2) Transcriptional activators of MDR induction in C. albicans. Initial studies in Saccharomyces cerevisiae have implicated two classes of transcriptional activators in azole-dependent MDR induction, PDR1/PDR3 and YAP1/YAP2. Candida homologues of these factors will be identified by PCR or expression in S. cerevisiae. Their role in MDR induction will be examined by gene disruption.