The role of ATO function in fungal pathogenesis

 描述(申请人提供)：白色念珠菌是发达国家最重要的真菌病原体，全世界每年死于念珠菌病的人数超过40万人，其中大约一半是由白色念珠菌引起的。播散性或侵袭性念珠菌病的风险最大的患者是那些天然免疫缺陷的患者，我们长期以来一直在研究白色念珠菌和巨噬细胞之间的动态和复杂的相互作用。吞噬作用刺激了一系列的转录和代谢变化，使细胞能够抵抗巨噬细胞施加的压力。这包括切换到糖异生生长模式，在这种模式下，细胞显然利用了各种不可发酵的碳源，我们已经证明，在动物模型中，吸收这些化合物所需的一些途径是完全毒力所必需的。我们的数据表明，氨基酸是吞噬小体中碳的特别重要来源。白色念珠菌利用氨基酸的分解代谢产生氨(来自氨基和侧链胺)，然后排泄到细胞外空间，在体外中和培养基和在体内中和吞噬小体。不能产生这种氨的菌株，例如缺乏Stp2的突变株，Stp2是一种调节氨基酸摄取和分解代谢的转录因子，它们占据了更酸性的吞噬小体，因此不能形成菌丝，更容易被巨噬细胞杀死。我们已经确定了一个被称为ATO的基因家族，即氨向外运输，相对于其他真菌，它在白色念珠菌中得到了极大的扩展(10个同源物，而酿酒酵母有3个同源物)。这十个基因中的许多，但不是全部，都是在吞噬细胞中诱导的，其中一组重叠的基因受到Stp2的调控。我们已经证明，ATO5的零突变或ATO1的显性点突变在体外和吞噬小体中损害碱化，并使细胞对巨噬细胞的杀伤略有敏感。我们认为这些表型是有限的，因为在这个大的基因家族中存在冗余的可能性，并且因为一些ATO蛋白专门用于在其他底物上的活性。事实上，我们已经发现了类似的碱化作用。 当细胞在N-乙酰氨基葡萄糖和羧酸(如α-酮戊二酸、丙酮酸和乳酸)上生长时，这些现象不受To1或To5突变的影响。我们还提出证据表明，在弱酸胁迫下，ATO蛋白需要维持胞浆pH的动态平衡。综上所述，我们假设ATO蛋白是醋酸盐和/或氨的向外(即胞浆到细胞外空间)的转运体，有助于在弱酸胁迫条件下(如吞噬小体)维持细胞的生理pH。因此，ATO对于白念珠菌在宿主中的适合性特别重要，这就是为什么这个家庭如此显著地扩大。我们将在两个特定的目标中进行测试，第一个目标是使用新的基因技术(Cas9/CRISPR系统)通过产生和测试多个突变体来探索ATO家族中的冗余和专门化。在第二个目标中，我们将使用pH敏感的GFP变体pHluorin来验证ATO是细胞pH动态平衡所必需的假设，评估有机酸的pH依赖毒性，并测试ATO缺陷菌株在输出醋酸盐或其他酸方面是否存在缺陷。总之，这些方法将是迄今为止对ATO之谜的最详细的分析--后生动物中不存在的一个保守的蛋白质家族，除了在宿主-病原体相互作用中的潜在作用外，几乎什么都不知道。

 DESCRIPTION (provided by applicant): Candida albicans is the most important fungal pathogen in the developed world, causing roughly half of the 400,000+ annual deaths attributed to candidiasis worldwide. The patients who are most at-risk for developing disseminated or invasive candidiasis are those with deficient innate immunity and we have long studied the dynamic and complex interaction between C. albicans and macrophages. Phagocytosis stimulates a dramatic program of transcriptional and metabolic changes that enable the cell to resist the stresses imposed by the macrophage. This includes a switch to a gluconeogenic growth mode in which the cell apparently utilizes a variety of nonfermentable carbon sources and we have shown that some of the pathways needed to assimilate these compounds are required for full virulence in animal models. Our data indicate that amino acids are particularly important sources of carbon in the phagosome. C. albicans uses the catabolism of amino acids to generate ammonia (derived from the amino and side chain amines) that is excreted into the extracellular space to neutralize the culture media in vitro and the phagosome in vivo. Strains unable to generate this ammonia, such as a mutant lacking Stp2, a transcription factor that regulates amino acid uptake and catabolism, occupy a more acidic phagosome and, as a result, fail to form hyphae and are more readily killed by the macrophage. We have identified a family of genes known as ATO, for Ammonia Transport Outward, that is greatly expanded in C. albicans relative to other fungi (ten homologs whereas Saccharomyces cerevisiae has three). Many, but not all, of the ten genes are induced in phagocytosed cells and an overlapping set are regulated by Stp2. We have shown that a null mutant of ATO5 or a dominant point mutant in ATO1 impairs alkalinization in vitro and in the phagosome, and renders the cell modestly more sensitive to killing by macrophages. We suggest that these phenotypes are limited because of the potential for redundancy in this large gene family, and because some of the ATO proteins are specialized for activity on other substrates. Indeed, we have identified similar alkalinization phenomena when cells are grown on N-acetylglucosamine and carboxylic acids such as α- ketoglutarate, pyruvate and lactate and these are not affected by ato1 or ato5 mutations. We also present evidence that ATO proteins are required to maintain cytosolic pH homeostasis under weak acid stresses. Together this leads us to hypothesize that the ATO proteins are outward (that is, cytosol to extracellular space) transporters of acetate and/or ammonia that help maintain physiological cellular pH in the weak acid stress conditions like that of the phagosome. Thus, the ATOs are particularly important for the fitness of C. albicans in the host, and this is why the family is so significantly expanded. We will test this in two specific aims, the first of which is to use novel genetic technologies (the Cas9/CRISPR system) to probe redundancy and specialization in the ATO family by generating and testing multiple mutants. In a second aim, we will test the hypothesis that the ATOs are required for cellular pH homeostasis using pHluorin, a pH-sensitive GFP variant, assessing pH-dependent toxicity of organic acids, and testing whether ato-deficient strains have defects in exporting acetate or other acids. Together, these approaches will be the most detailed analysis yet of the ATO enigma - a conserved protein family that is absent in metazoans, about which virtually nothing is known beyond a potential role in host-pathogen interactions.