The role of ATO function in fungal pathogenesis

 描述（由申请人提供）：白色念珠菌是发达国家最重要的真菌病原体，全球每年因念珠菌病死亡的40万多人中约有一半是由白色念珠菌引起的。最有可能发展为播散性或侵袭性念珠菌病的患者是先天免疫缺陷的患者，我们长期以来一直研究了C。白色念珠菌和巨噬细胞。吞噬作用刺激转录和代谢变化的戏剧性程序，使细胞能够抵抗巨噬细胞施加的压力。这包括切换到一个产毒的生长模式，其中细胞显然利用各种不可发酵的碳源，我们已经表明，同化这些化合物所需的一些途径是在动物模型中完全毒力所必需的。我们的数据表明，氨基酸是吞噬体中特别重要的碳源。C.白色念珠菌使用氨基酸的催化剂来产生氨（来源于氨基和侧链胺），所述氨被分泌到细胞外空间中以在体外中和培养基和在体内中和吞噬体。不能产生这种氨的菌株，如缺乏Stp 2的突变体，Stp 2是一种调节氨基酸摄取和catalysts的转录因子，占据了酸性更强的吞噬体，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，即氨转运蛋白，它在C.白色念珠菌相对于其他真菌（10个同源物，而酿酒酵母有3个）。许多，但不是所有的，十个基因被诱导吞噬细胞和重叠的一套由Stp 2调节。我们已经表明，一个无效突变的ATO 5或一个显性点突变的ATO 1损害碱化在体外和吞噬体，并使细胞适度更敏感的巨噬细胞的杀伤。我们认为，这些表型是有限的，因为在这个大的基因家族的冗余的潜力，因为一些ATO蛋白是专门为其他底物的活动。事实上，我们已经发现了类似的碱化 当细胞在N-乙酰葡糖胺和羧酸如α-酮戊二酸、丙酮酸和乳酸上生长时，这些现象不受ato 1或ato 5突变的影响。我们还提出的证据表明，ATO蛋白是必需的，以维持细胞溶质pH值的稳态下，弱酸应力。总之，这导致我们假设ATO蛋白是乙酸盐和/或氨的向外（即，胞质到细胞外空间）转运蛋白，其有助于在弱酸应激条件下（如吞噬体的条件下）维持生理细胞pH。因此，ATO对C.白念珠菌在宿主中，这就是为什么家庭如此显着扩大。我们将在两个具体目标中测试这一点，第一个是使用新的遗传技术（Cas9/CRISPR系统）通过产生和测试多个突变体来探测ATO家族中的冗余和专业化。在第二个目标中，我们将使用pH荧光蛋白（一种pH敏感的GFP变体）测试ATO是细胞pH稳态所需的假设，评估有机酸的pH依赖性毒性，并测试ATO缺陷菌株是否在输出乙酸盐或其他酸方面存在缺陷。总之，这些方法将是对ATO之谜的最详细的分析-一种在后生动物中不存在的保守蛋白质家族，除了在宿主-病原体相互作用中的潜在作用之外，几乎一无所知。

 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.