ANTIFUNGAL RESPONSE--MDR INDUCTION

真菌可能以特定的方式对抗真菌化疗的压力作出反应，最终降低治疗的疗效。这种“抗真菌反应”类似于热休克反应，但在机制上不同。本提案的重点是一种抗真菌反应，称为多药耐药诱导，涉及编码多药耐药（MDR）转运蛋白的基因的快速药物依赖性转录诱导。具体来说，将白色念珠菌、克鲁氏念珠菌或酿酒酵母暴露于某些药物（包括抗真菌唑咪康唑和克霉唑、苯并咪唑阿苯达唑和磺胺嘧啶）15-20分钟可诱导编码MDR特异性转运体的mrna增加3 - 10倍。最初的研究表明，MDR诱导是念珠菌对苯并咪唑的内在抗性以及诱导MDR的化合物（阿苯达唑和磺胺嘧啶）与作为MDR转运体底物的化合物（唑）之间拮抗活性的最可能解释。假设，MDR诱导也可能有助于对唑类的表型适应（反映在“尾随效应”上），不同真菌对不同唑类的不同敏感性，以及获得性抗真菌耐药性。本提案的具体目的是检查：(1)MDR诱导的频谱。新的抗真菌药物和抗感染药物通常用于免疫功能低下的患者将测试白色念珠菌耐多药诱导和拮抗唑活性。将比较对唑敏感和耐药菌株以及白色念珠菌酵母菌和菌丝形式的耐多药诱导和拮抗作用。从光秃念珠菌、烟曲霉和新型隐球菌中扩增MDR基因片段，作为这些真菌诱导MDR研究的探针。(2)白色念珠菌耐多药诱导的转录激活因子。在酿酒酵母中进行的初步研究表明，两类转录激活因子PDR1/PDR3和YAP1/YAP2参与了唑依赖性MDR诱导。这些因子的假丝酵母同源物将通过PCR或酿酒葡萄球菌的表达进行鉴定。它们在耐多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.