Echinocandin resistant & hypersensitive C. albicans

描述(申请人提供)：白色念珠菌是最常引起人类侵袭性疾病的真菌。这种感染具有严重后遗症和死亡的高风险，随着对危及患者免疫防御的严重疾病的治疗取得进展，易受感染的个人数量继续上升。在这种真菌的血液感染中，可归因性死亡率接近40%。侵袭性念珠菌感染死亡率高的原因之一是可用于治疗的抗真菌药物选择有限。棘球菌素是最近临床上可用的抗真菌药物的补充，具有几个重要的优点。为了保持棘球菌素在未来潜在的广泛使用中的效用，及早识别导致棘球绦虫耐药性的突变将是有用的。棘球菌素的靶标是产生念珠菌细胞壁的主要成分的酶。完整的细胞壁对真菌细胞的生存至关重要。在模式真菌酿酒酵母中，已经确定了许多调控细胞壁合成活动的调节电路。对棘球菌素耐药或高度敏感的酵母菌突变体为分析这些调控回路提供了一个切入点。尽管从酿酒酵母中获得的洞察力可以学到很多东西，但白色念珠菌的生理和基因组显示出与酵母菌的重要区别，因为它已经进化到针对宿主的免疫系统保持细胞壁。我们建议对白色念珠菌本身对棘球菌素的耐药性和超敏反应进行基因组调查。一组杂合的白色念珠菌突变株将在棘球绦虫存在的情况下进行生长筛选。使用转座子产生突变体，转座子以最大随机的方式插入，仅使用二核苷酸作为识别序列。通过我们在中试筛选中开发的一种方案，将收集到抗性和过敏性突变。他们的表型将得到确认，转座子插入的位置将被确定。在野生型菌株中，转座子插入与棘球绦虫相关表型的联系将通过在相同的基因座上重新产生突变来确定。由此产生的对念珠菌棘球菌素反应基因的概述将提供探针，以剖析调节这种病原体维持细胞壁完整性的能力的网络。

DESCRIPTION (provided by applicant): Candida albicans is the fungus most often causing invasive disease in humans. The number of susceptible individuals to this infection, which carries a high risk of serious sequelae and death, continues to rise, in parallel with the advances in treatment of serious illnesses that compromise patients' immune defenses. In bloodstream infections with this fungus, attributable mortality is close to 40%. One reason for the high mortality of invasive Candida infection is the limited choice of antifungal drugs available for its treatment. Echinocandins have been a recent addition to the clinically available antifungals, with several important advantages. In order to preserve the utility of the echinocandins during potential future widespread use, early identification of mutations which give rise to echinocandin resistance will be useful. The target of echinocandins is the enzyme that produces the major component of Candida's cell wall. An intact wall is critical for the fungal cell's survival. In the model fungus Saccharomyces cerevisiae, numerous regulatory circuits have been identified which govern the activity of cell wall synthesis. Saccharomyces mutants which are resistant or hypersensitive to echinocandins have provided an entry point for the analysis of these regulatory circuits. Though much can be learned from the insights gained in S. cerevisiae, the physiology and the genome of C. albicans show important differences from Saccharomyces, because it has evolved to maintain its cell wall against the host's immune system. We propose a genomic survey of resistance and hypersensitivity to echinocandins in C. albicans itself. A collection of heterozygous C. albicans mutants will be screened for their growth in the presence of an echinocandin. The mutants are generated using a transposon which inserts in a maximally random fashion, using only a dinucleotide as a recognition sequence. By a protocol we developed in a pilot screen, resistant and hypersensitive mutants will be collected. Their phenotypes will be confirmed and the locus of the transposon insertion will be identified. Linkage of the transposon insertion to the echinocandin-related phenotype will be determined by recreating a mutation in the same locus, in the wild type strain. The resulting overview of genes involved in Candida's echinocandin response will provide probes to dissect the networks that regulate this pathogen's ability to maintain the integrity of its cell wall.