Glucan Binding to Azole Drugs: A Novel Resistance Mechanism in Candida albicans

描述(申请人提供)：致病酵母白色念珠菌(钙)是一种常见的原因，口腔和全身真菌感染的免疫功能低下的人和阴道感染的妇女。念珠菌感染通常用唑类药物治疗，在这些患者群体中经常出现唑类耐药。唑类耐药的主要机制包括外排泵表达增加和麦角甾醇生物合成过程中酶的改变。然而，在许多耐药的临床分离株中，这些耐药机制尚未被确定。最近，在念珠菌生物被膜中发现了第三种耐唑类药物的机制--形成在粘膜表面和植入医疗器械上的细胞和细胞外物质的基质。这些生物膜中高水平的唑类耐药性是由于与唑类药物结合的细胞壁中(-1，3葡聚糖)的产生增加所致。我们的假设是，咪唑类化合物的(-1，3-葡聚糖结合)不仅在生物膜中是耐药成分，而且在浮游临床耐药菌株中也是耐药成分。偶氮类药物与细胞壁的葡聚糖结合会阻止药物进入细胞质，从而增加耐药性。总体目标是评估与(-1，3-葡聚糖)的结合对念珠菌耐药的贡献。具体地说，这项建议调查了(-1，3葡聚糖结合)如何影响真菌细胞中的药物积累，并评估了(-1，3葡聚糖结合)在一组临床分离株中的结合，包括尚未知耐药机制的分离株。本应用的具体目的是：1.确定(-1，3)葡聚糖结合对氟康唑在白色念珠菌中积累的影响。真菌细胞的唑类积累可能是几个相互竞争的过程的结果，包括输入和外流(内部积累)，以及可能的(-1，3葡聚糖结合(外部积累)。放射性标记的FLC在细胞中的累积将在改变细胞中葡聚糖含量的条件下进行评估。2.评估药物敏感性改变的临床分离株中(-1，3-葡聚糖结合)。在尚未确定耐药机制的耐药临床分离株中，将评估唑与(-1，3葡聚糖)的结合和细胞中(-1，3葡聚糖)的水平。在可预见的未来，唑类药物和真菌细胞之间的相互作用将继续是临床上重要的问题。(-1，3葡聚糖与唑类药物的结合是这些相互作用的一个新的和重要的方面。对这一过程的全面描述，加上我们对药物/细胞相互作用的其他方面的理解，有可能有助于改进真菌感染和耐药性的诊断、治疗和预防。致病酵母白色念珠菌会在口腔、阴道和血液中引起重大的人类疾病。这项建议将分析抗真菌药物氟康唑和白色念珠菌之间的相互作用。特别是，它将检验真菌细胞壁充当海绵的假设，吸收药物，从而使细胞在药物存在的情况下保持不变。

DESCRIPTION (provided by applicant): The pathogenic yeast Candida albicans (Ca) is a frequent cause of oral and systemic fungal infections in immune-compromised people and of vaginal infections in women. Candida infections are usually treated with azole drugs, and azole resistance arises frequently in these patient populations. The major mechanisms of azole resistance include increased expression of efflux pumps and alterations in enzymes in ergosterol biosynthesis. However, these mechanisms of resistance have not been identified in many resistant clinical isolates. Recently, a third mechanism of azole resistance has been found in Candida biofilms - matrices of cells and extracellular material that forms on mucosal surfaces and implanted medical devices. The high levels of azole resistance in these biofilms is due to increased production of (-1,3 glucan in the cell wall that binds to azole drugs. Our Hypothesis is that (-1,3 glucan binding of azoles is a component of resistance not only in biofilms, but in planktonic clinically resistant isolates. Glucan binding of azoles to the cell wall would prevent the drug from reaching the cytoplasm, increasing resistance. The Overall Goal is to evaluate the contribution of azole binding to (-1,3 glucan as a component of drug resistance in Candida. Specifically, this proposal investigates how (-1,3 glucan binding affects drug accumulation in fungal cells, and it evaluates (-1,3 glucan binding in a collection of clinical isolates, including isolates with no known resistance mechanisms. The Specific Aims of this application are: 1. To determine the effect of (-1,3 glucan binding on fluconazole accumulation in Candida albicans. Azole accumulation by fungal cells is likely to be the result of several competing processes, including import and efflux (internal accumulation), and possibly (-1,3 glucan binding (external accumulation). Radiolabeled FLC accumulation in cells will be evaluated under conditions that alter the glucan content of the cells. 2. To assess (-1,3 glucan binding in clinical isolates with altered drug susceptibilities. Azole binding to (-1,3 glucans, and (-1,3 glucan levels in the cells will be assessed in resistant clinical isolates in which no known mechanism of resistance has been identified. The interactions between azoles and fungal cells will continue to be clinically significant issues for the foreseeable future. (-1,3 glucan binding of azole drugs is a new and important aspect of these interactions. A full characterization of this process, together with our understanding of the other facets of drug/cell interactions, has the potential to contribute to improvements in diagnosis, treatment and prevention of fungal infections and of resistance. PUBLIC HEALTH RELEVANCE The pathogenic yeast Candida albicans causes significant human disease in the mouth, vagina and blood stream. This proposal will analyze the interaction between the antifungal drug fluconazole and C. albicans. In particular, it will test the hypothesis that the fungal cell wall acts as a sponge, soaking up the drug, thus allowing the cells to persist in the presence of drug.