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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：白色念珠菌是最重要的人相关真菌，既是微生物群的组成部分，也是多器官系统的病原体。白色念珠菌引起感染，从易于治疗的粘膜感染（鹅口疮，阴道）到难治性和经常致命的全身感染。传播感染通常是固有免疫力缺陷的结果，这融合了白色念珠菌 - 表现细胞相互作用的研究。巨噬细胞的吞噬作用会在真菌中引起动态和复杂的反应，包括动态代谢转移到谷胱甘肽生长模式，我们已经表明，这种转移的许多方面是完全病毒所必需的。在体外对该系统进行建模已经发现了该生物体从根本上改变细胞外pH的能力，作为这些代谢变化的副产品。预计氨基酸在宿主中有很多，并且它们用于满足细胞碳需求的用途会导致细胞外pH的急剧上升，这是由于氨的出口驱动，也许是其他基本的氮化合物。我们提出了一个模型，其中该氨源自氨基酸的氨基或侧链胺作为代谢副产品。虽然这是大多数生物体氨酸分解代谢的潜在结果，但念珠菌（尤其是白色念珠菌）的pH变化比以前描述的要强大得多。理解这一过程是我对这种生物如何适应基本代谢功能以将其作为成功的共生和病原体支持的长期兴趣的一部分。遗传分析已经鉴定出非卵巢化突变体，这些突变体富含氨基酸进口和分解代谢功能。最严重缺陷的突变体是在STP2中，编码调节氨基酸渗透酶表达的转录。在与宿主细胞接触期间，缺乏STP2的突变体受到损害：它们发芽较差，并且比野生型对照组更容易被巨噬细胞杀死，对巨噬细胞造成的损害更少。此外，在酸性的吞噬肿瘤体中发现了STP2δ细胞，以及杀死的细胞。相反，野生型细胞占据了更中性的吞噬体，表明它们具有改变STP2δ细胞缺乏细胞内pH的机制。其他人则表明，STP2δ突变体在念珠菌病的蝇模型中被减弱。编码ER链酮的另外两个非静碱化突变体CSH3Δ和编码氨基酸分解代谢酶尿素胺胺的dur1,2δ在小鼠中是无毒性的。这是有很大的初步证据表明，调节碱化的因素也改变了宿主 - 病原体的相互作用，尽管两者之间的精确连接尚未牢固确定。最近，我们在TCA循环的二羧酸中间体生长过程中发现了第二种碱化机制，并将表征该机制。该应用提案以检验以下假设：关键宿主壁ni的碱化（包括吞噬体）通过抑制宿主的免疫反应来促进白色念珠菌的病毒。我们将通过测试模型的代谢适应性来做到这一点，这些适应使白色念珠菌中的这种现象比其他真菌更加有活力，包括确定释放氨（和其他潜在的碱性代谢产物）的来源。我们还将确定碱化是否发生在吞噬细胞内，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。