Extracellular pH modulation by Candida albicans in vitro and in vivo

描述(申请人提供)：白色念珠菌是最重要的人类相关真菌，既作为微生物区系的组成部分存在，也作为多器官系统的病原体存在。白念珠菌引起各种感染，从容易治疗的粘膜感染(鹅口疮、阴道炎)到难治性的、往往是致命的全身感染。已传播 感染往往是先天免疫缺陷的结果，这促使人们研究白色念珠菌与吞噬细胞的相互作用。巨噬细胞的吞噬作用在真菌中诱导了动态和复杂的反应，包括戏剧性的代谢转变为糖异生生长模式，我们已经证明这种转变的许多方面是完全毒力所必需的。在体外对这个系统进行建模，揭示了作为这些代谢变化的副产品，这种有机体从根本上改变细胞外pH的能力，这是以前未知的能力。据预测，宿主体内含有丰富的氨基酸，它们用于满足细胞碳需求导致胞外pH急剧上升，这是由于氨的输出，也许还有其他碱性含氮化合物的输出。我们提出了一个模型，在这个模型中，这种氨来自氨基酸的氨基或侧链氨基，作为代谢的副产品。虽然这是大多数生物体中氨基酸分解代谢的潜在后果，但念珠菌(特别是白色念珠菌)表现出比以前描述的更强烈的pH变化。了解这一过程是我长期感兴趣的一部分，我一直感兴趣的是这种有机体如何适应基本的代谢功能，以支持它作为成功的共生和病原体。遗传分析已经确定了非碱化突变体，它们富含氨基酸输入和分解代谢功能。最严重的缺陷突变是STP2，它编码一种转录，调节氨基酸通透酶的表达。缺乏STP2的突变体在与宿主细胞接触时会受到损害：它们发芽能力差，而且比野生型对照更容易被巨噬细胞杀死，对巨噬细胞造成的损害更小。此外，stp2？细胞存在于酸性吞噬小体中，被杀死的细胞也是如此。相反，野生型细胞占据一个更中性的吞噬酶体，这表明它们有一种机制来改变细胞内缺乏stp2？细胞。其他人已经证明了stp2？突变株在假丝酵母菌病的苍蝇模型中减弱；另外两个非碱化突变体csh3？，编码氨基酸渗透的ER伴侣，和du1，2？，编码氨基酸分解酶尿素氨解酶，在小鼠中是无毒的。因此，有强有力的初步证据表明，调节碱化的因素也会改变宿主-病原体的相互作用，尽管两者之间的确切联系尚未得到确定。我们最近发现了在TCA循环的二元酸中间体上生长过程中的第二种碱化机制，并将对该机制进行表征。这项应用提出了一种假设，即包括吞噬酶体在内的关键宿主生态位的碱化通过抑制宿主免疫反应来促进白念珠菌的毒力。我们将通过测试我们的模型来测试代谢适应，使这种现象在白色念珠菌中比在其他真菌中更活跃，包括确定释放的氨(和其他潜在的基本代谢物)的来源。我们还将确定吞噬细胞内是否发生碱化，这是否是诱导菌丝形态发生的关键信号。最后，我们将使用活细胞成像来剖析白念珠菌在巨噬细胞中的细胞内运输，并确定碱化和氨释放在调节内吞成熟中的作用。

DESCRIPTION (provided by applicant): Candida albicans is the most important human-associated fungus, existing as both an integral component of the microbiota and as a pathogen of multiple organ systems. C. albicans causes infections ranging from easily treatable mucosal infections (thrush, vaginitis) to refractory and frequently fatal systemic infections. Disseminated infections are often a result of defects in innate immunity and this has motivated studies of C. albicans-phagocyte interactions. Phagocytosis by macrophages induces a dynamic and complex response in the fungus, including a dramatic metabolic shift to a gluconeogenic mode of growth, and we have shown that many aspects of this shift are required for full virulence. Modeling this system in vitro has uncovered a previously unknown ability of this organism to radically change extracellular pH as a byproduct of these metabolic changes. Amino acids are predicted to be plentiful in the host, and their use to satisfy cellular carbon requirements result in a dramatic rise in extracellular pH, driven by the export of ammonia, and perhaps other basic nitrogenous compounds. We propose a model in which this ammonia derives from the amino or side-chain amine groups of the amino acids as a metabolic byproduct. While this is a potential consequence of amino acid catabolism in most organisms, Candida species (C. albicans in particular) show a far more robust pH change than has been previously described. Understanding this process is part of my long-standing interest in how this organism has adapted basic metabolic functions to support it as a successful commensal and pathogen. Genetic analysis has identified non-alkalinizing mutants, which are enriched for amino acid import and catabolic functions. The most severely deficient mutant is in STP2, encoding a transcription that regulates amino acid permease expression. Mutants lacking STP2 are impaired during contact with host cells: they germinate poorly and are both more susceptible to killing by macrophages and cause less damage to macrophages than wild-type controls. Moreover, stp2? cells are found in acidic phagolysosomes, as are killed cells. In contrast, wild-type cells occupy a more neutral phagolysosome, suggesting they have a mechanism to alter intracellular pH lacking in stp2? cells. Others have shown that stp2? mutants are attenuated in a fly model of candidiasis; two other non-alkalinizing mutant, csh3?, encoding an ER chaperone for amino acid permeases, and dur1,2?, encoding the amino acid catabolic enzyme urea amidolyase, are avirulent in mice. Thus, there is strong preliminary evidence that factors that regulate alkalinization also alter the host-pathogen interaction, though the precise connection between the two has not been firmly established. We have recently discovered a second alkalinization mechanism during growth on dicarboxylic acid intermediates of the TCA cycle, and will characterize this mechanism as well. This application proposes to test the hypothesis that alkalinization of key host niches, including the phagolysosome, promotes virulence of C. albicans by inhibiting the host immune response. We will do so by testing our model for the metabolic adaptations that make this phenomenon much more vigorous in C. albicans than in other fungi, including determining the source of the ammonia (and other potential basic metabolites) released. We will also determine whether alkalinization occurs within the phagocyte and if this is the key signal that induces hyphal morphogenesis. Finally, we will use live cell imaging to dissect the intracellular trafficking of C. albicans in the macrophage and determine the role of alkalinization and ammonia release in modulating endocytic maturation.