Alpha-(1,3)-Glucan as a Target for Antifungal Therapy

细菌细胞壁是抗感染药物的传统靶点，但直到最近才开发出类似的抗真菌药物（棘白菌素）。虽然很有前途，但棘白菌素对于不严重依赖β-（1，3）-葡聚糖来保持细胞完整性的真菌的适用性有限。一个类似但完全未探索的目标是α-（1，3）-葡聚糖，这是除念珠菌属以外的所有真菌病原体细胞壁的结构特征。由于缺乏这种多糖的突变体是无毒的，但可行的，正向遗传学成为一种手段，以确定候选药物靶点，它也更容易研究所发现的基因的作用和调控。在本研究计划中，我们将开始检验以下假设：参与α-（1，3）-葡聚糖合成、加工和调节的基因和基因产物可作为抗真菌剂的选择性靶点。作为我们的模型系统，我们将使用H。荚膜多糖，其中α-（1，3）-葡聚糖在毒力和生物学中的作用已得到最彻底的研究。我们的具体目标是： I.确定与α-（1，3）-葡聚糖生产有关的基因。H.将筛选通过农杆菌介导的诱变产生的荚膜突变体的α-（1，3）-葡聚糖缺陷或AGS 1（其编码α-（1，3）-葡聚糖合酶）调节的丧失。将回收所有这些突变体中插入侧翼的DNA并测序，并从测序的组织胞浆菌基因组中鉴定中断的基因。选择用于进一步研究的基因将是那些在各种其他真菌中保守的基因。 二.鉴定和表征与α-（1，3）-葡聚糖产生有关的基因。对于每一个感兴趣的突变体，我们将通过以下方式验证该突变对表型负责：（i）用野生型基因补充突变体;或（ii）使用RNAi使野生型菌株中的该基因沉默。将在培养的巨噬细胞和肺部感染的小鼠模型中评价突变体的相对毒力。将选择具有最高潜力作为药物靶标的基因，使用确定的突变体和报告基因构建体进行基因功能和表达的更详细分析。

The bacterial cell wall is a traditional target for anti-infective drugs, but only recently have analogous antifungal drugs (echinocandins) been developed. While promising, echinocandins have limited applicability for fungi that do not rely heavily on beta-(1,3)-glucan for cell integrity. A similar but completely unexplored target is alpha-(1,3)-glucan, a structural feature of the cell wall of all fungal pathogens other than Candida spp. Since mutants lacking this polysaccharide are avirulent but viable, forward genetics becomes a means to identify candidate drug targets, and it is also far easier to study the role and regulation of the discovered genes. In this research plan, we will begin to test the hypothesis that genes and gene products involved in the synthesis, processing, and regulation of alpha-(1,3)-glucan can serve as selective targets for antifungal agents. As our model system, we will use H. capsulatum, in which the role of alpha-(1,3)-glucan in virulence and biology has been most thoroughly studied. Our specific aims are to: I. Identify genes involved in the production of alpha-(1,3)-glucan. H. capsulatum mutants, generated by Agrobacterium-mediated mutagenesis, will be screened for deficiency of alpha-(1,3)-glucan or for loss of regulation of AGS1 (which encodes alpha-(1,3)-glucan synthase). DNA flanking the insertions in all of these mutants will be recovered and sequenced, and the interrupted genes will be identified from the sequenced Histoplasma genome. Genes selected for further study will be those that are conserved among a wide variety of other fungi. II. Validate and characterize genes implicated in production of alpha-(1,3)-glucan. For each of the mutants of interest, we will validate that the mutation is responsible for the phenotype by either (i) complementing the mutant with the wild-type gene the mutation; or (ii) using RNAi to silence that gene in a wild-type strain. The mutants will be evaluated for relative virulence in cultured macrophages and in a mouse model of pulmonary infection. Genes with the highest potential as drug targets will be selected for more detailed analysis of gene function and expression, using defined mutants and reporter gene constructs.