Extracellular pH modulation by Candida albicans in vitro and in vivo

白色念珠菌对细胞外 pH 值的体外和体内调节

• 批准号: 8847274
• 负责人: Michael C Lorenz
• 金额: $ 44.32万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8847274
• 关键词: Acetates; Affect; Alkalinization; Amines; Amino Acid Permease; Amino Acids; Ammonia; Animal Model; Animals; Antifungal Agents; Assimilations; Attenuated; Biochemical; Biological Assay; Biological Models; Candida; Candida albicans; Candidiasis; Carbon; Catabolism; Cells; Citric Acid Cycle; Complex; Culture Media; Data; Defect; Development; Diagnostic; Dicarboxylic Acids; Disease Progression; Employee Strikes; Environment; Enzymes; Epidemiology; Excretory function; Extracellular Space; Fatty Acids; Gastrointestinal tract structure; Generations; Genes; Genetic; Genetic Transcription; Genomics; Germination; Goals; Growth; Health; Human; Immune response; Immune system; Immunocompromised Host; In Vitro; Infection; Laboratories; Life; Metabolic; Metabolic Pathway; Metabolism; Microbe; Modeling; Molecular Chaperones; Morbidity - disease rate; Morphogenesis; Mus; Mutation; Mycoses; Natural Immunity; Nitrogen; Nucleotides; Nutrient; Oral candidiasis; Organism; Pathogenicity; Pathway interactions; Phagocytes; Phagocytosis; Phagolysosome; Population; Process; Publishing; Reaction; Refractory; Regulation; Role; Sepsis; Side; Signal Transduction; Source; Starvation; Stress; Systemic infection; Testing; Therapeutic; Toxin; Urea; Vaginitis; Virulence; Virulence Factors; Work; Yeasts; base; body system; capsule; catalase; cell killing; cell type; cellular imaging; extracellular; fly; fungus; genetic analysis; genetic approach; in vivo; interdisciplinary approach; interest; killings; macrophage; mortality; mutant; pathogen; programs; research study; response; trafficking; trait; transcription factor

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, stp2Δ 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 stp2Δ cells. Others have shown that stp2Δ mutants are attenuated in a fly model of candidiasis; two other non-alkalinizing mutant, csh3Δ, encoding an ER chaperone for amino acid permeases, and dur1,2Δ, 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.

描述（由申请人提供）：白色念珠菌是最重要的人类相关真菌，既作为微生物群的组成部分又作为多个器官系统的病原体而存在。白色念珠菌引起的感染范围从容易治疗的粘膜感染（鹅口疮、阴道炎）到难治性且常常致命的全身感染。传播 感染通常是先天免疫缺陷的结果，这激发了对白色念珠菌-吞噬细胞相互作用的研究。巨噬细胞的吞噬作用在真菌中诱导动态且复杂的反应，包括向糖异生生长模式的急剧代谢转变，并且我们已经证明这种转变的许多方面对于完全毒力是必需的。对该系统的体外建模揭示了该生物体具有一种以前未知的能力，可以从根本上改变细胞外 pH 值，作为这些代谢变化的副产品。预计宿主体内氨基酸含量丰富，并且利用它们来满足细胞碳需求会导致细胞外 pH 值急剧上升，这是由氨和其他碱性含氮化合物的输出驱动的。我们提出了一个模型，其中氨源自氨基酸的氨基或侧链胺基作为代谢副产物。虽然这是大多数生物体中氨基酸分解代谢的潜在后果，但念珠菌属物种（特别是白色念珠菌）表现出比之前描述的更剧烈的 pH 变化。了解这个过程是我长期以来对这种生物体如何适应基本代谢功能以支持其作为成功的共生体和病原体的兴趣的一部分。遗传分析已鉴定出非碱化突变体，这些突变体富含氨基酸输入和分解代谢功能。最严重缺陷的突变体是STP2，编码调节氨基酸通透酶表达的转录。缺乏STP2的突变体在与宿主细胞接触过程中会受到损害：它们发芽能力较差，并且更容易被巨噬细胞杀死，并且对巨噬细胞造成的损害比野生型对照要小。此外，stp2Δ细胞与被杀死的细胞一样存在于酸性吞噬溶酶体中。相比之下，野生型细胞占据更中性的吞噬溶酶体，这表明它们具有改变 stp2Δ 细胞所缺乏的细胞内 pH 值的机制。其他研究表明，stp2Δ 突变体在念珠菌病果蝇模型中减弱；另外两种非碱化突变体 csh3Δ（编码氨基酸渗透酶的 ER 伴侣）和 dur1,2Δ（编码氨基酸分解代谢酶尿素酰胺裂解酶）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与病原体的相互作用，尽管两者之间的精确联系尚未牢固确立。我们最近发现了 TCA 循环的二羧酸中间体生长过程中的第二种碱化机制，并将表征该机制。本申请旨在测试以下假设：包括吞噬溶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色念珠菌的毒力。我们将通过测试我们的代谢适应模型来做到这一点，这些代谢适应使这种现象在白色念珠菌中比在其他真菌中更加强烈，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，以及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色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。