DNA Repair Genes and Acquired Drug Resistance in Candida

描述(申请人提供)：白色念珠菌是人类最常见的真菌病原体。通常情况下，当宿主变得虚弱或免疫抑制时，可能会导致共生念珠菌病。在血源性播散性感染中，即使使用各种抗真菌药物治疗，死亡率也可能达到50%。获得性抗真菌耐药性正成为一个严重的临床问题，但耐药性产生的机制尚不清楚。导致耐药的基因改变可能涉及突变、纯合子、染色体易位和重复序列的改变。我们建议研究白念珠菌同源基因在一些DNA修复途径中的作用，以确定导致念珠菌耐药的基因组变化所需的那些基因。 我们将构建在每个主要DNA修复过程-碱基切除修复、核苷酸切除修复、错配修复和双链断裂修复-中涉及的两个基因副本均为零突变的菌株。我们将使用一系列分析来表征每个缺失突变体的DNA修复活性。我们还将确定各种突变菌株对常用抗真菌药物获得耐药性的速度，以及在耐药菌株中发现的病变类型，以确定哪些DNA修复过程在念珠菌的耐药性发展中具有直接作用。 这些实验的结果将使我们能够确定抗真菌耐药表型产生的方式，并建议联合治疗的靶点，从而大大减少获得性耐药问题。我们的数据还可能解决体内观察到的白色念珠菌显著基因组不稳定性的潜在机制。核型重排在实验室中相对较少发生，但在临床分离株中非常常见。到目前为止，假丝酵母菌还没有试图对这些观察到的机制进行严格的分析。由于我们预计我们将在我们分离的耐药突变株中发现改变的核型，我们拟议的实验将指向其中一些机制，为未来关于耐药性、念珠菌基因组稳定性和相关过程的工作提供一个坚实的框架。

DESCRIPTION (provided by applicant): Candida albicans is the most common fungal pathogen of humans. Normally a commensal, candidiasis may result when the host becomes debilitated or immunosuppressed. In hematogenously disseminated infections, mortality can reach 50%, even with treatment using various antifungal drugs. Acquired antifungal drug resistance is becoming a serious clinical problem, but the mechanisms by which resistance develops are unknown. The genetic changes leading to drug resistance may involve mutation, homozygosis, chromosome translocations, and alterations of repeat sequences. We propose to examine the roles of C. albicans homologs of genes known to be involved in a number of DNA repair pathways, to identify those genes that are required for the genomic changes resulting in antifungal drug resistance in Candida. We will construct strains with null mutations in both copies of genes involved in each of the primary DNA repair processes - base excision repair, nucleotide excision repair, mismatch repair and double-strand break repair. We will use a series of assays to characterize the DNA repair activities of each of the deletion mutants. We will also determine the rate at which the various mutant strains acquire resistance to commonly used antifungal agents, and the type of lesions found in the resistant strains, to identify those DNA repair processes having direct roles in drug resistance development in Candida. The results of these experiments will allow us to determine the ways in which antifungal resistance phenotypes arise and suggest targets for combined therapy that would greatly decrease the problem of acquired resistance. Our data may also address the mechanisms underlying the striking genomic instability of C. albicans observed in vivo. Karyotypic rearrangements arise relatively rarely in the laboratory but are very common in clinical isolates. To date, a rigorous analysis of the mechanisms underlying these observations has not been attempted in Candida. Since we expect that we will find altered karyotypes among the drug-resistant mutants we isolate, our proposed experiments will point to some of these mechanisms, providing a solid framework for future work on drug resistance, Candida genome stability, and associated processes.