Metal nutrients and metallophore-like molecules for a fungal pathogen

真菌病原体的金属营养物和类金属载体分子

• 批准号: 10231544
• 负责人: Valeria C Culotta
• 金额: $ 24.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231544
• 关键词: Affinity; Anabolism; Animals; Bacteria; Binding; Biochemical; Biological Assay; Biology; Candida; Candida albicans; Candidiasis; Cells; Chemicals; Chemistry; Collaborations; Complex; Electron Nuclear Double Resonance; Elements; Environment; Exhibits; Face; Generations; Growth; Homeostasis; Human; Infection; Innate Immune Response; Invaded; Ions; Life; Mass Spectrum Analysis; Metals; Methods; Microbe; Micronutrients; Molecular Sieve Chromatography; Molecular Weight; Nature; Nutrient; Nutritional Immunity; Organism; Pathogenesis; Pathway interactions; Process; Production; Property; Public Health; Research; Role; Siderophores; Site; Spectrum Analysis; Starvation; Stress; Surveys; Testing; Time; Transition Elements; Virulence; Washington; Yeasts; experimental study; extracellular; fungus; insight; metal metabolism; metalloenzyme; mouse model; mutant; pathogen; pathogenic fungus; pathogenic microbe; programs; success; uptake; virtual

Transition metals including Cu, Mn, Zn and Fe are essential nutrients for all life forms. Microbial pathogens face challenges in acquiring these vital elements, as the host deliberately attempts to starve microbes of their metals through processes collectively known as nutritional immunity. Successful pathogens have evolved elaborate methods to evade nutritional immunity and capture metals from their host; one mechanism involves secretion of ‘metallophores”. Metallophores are small (≤1.5 kDa) molecules that bind extracellular metals with high affinity and deliver the ion to the microbe for uptake. Bacteria secrete various metallophores for Fe, Cu or Zn, although none have been identified for Mn. Pathogenic fungi are thought to only secrete Fe-metallophores and only in certain species. Candida albicans is an opportunistic fungal pathogen believed to not produce metallophores of any kind, although the yeast is dependent on host metals for pathogenesis. Recently, our lab has challenged the dogma of no metallophores for Candida sp and obtained evidence that C. albicans does in fact produce metallophore-like compounds that exhibit strong selectivity for binding Mn2+ or Cu2+. We call these Mn-MBC and Cu-MBC for metal binding complex. The metal coordination sites of Mn-MBC and Cu-MBC are distinct, as are their chromatographic properties, indicating that Mn-MBC and Cu-MBC are different molecules. Interestingly, production of Cu-MBC, but not Mn-MBC, is induced by Fe, indicating a role for Cu- MBC in Fe homeostasis. Currently we do not know the chemical nature of the MBC molecules, their occurrence among diverse fungi, or their roles in metabolism of metals for fungal pathogenesis. Over this two- year research plan, we shall test for MBC production from fungi outside of Candida sp; we will chemically identify C. albicans Mn-MBC and Cu-MBC, and will begin to probe their mechanism of action. Aim 1: To survey MBC across diverse fungi and chemically identify the molecules from C. albicans. Using a size exclusion chromatography/ICP-mass spectrometry/ENDOR spectroscopy approach that we have developed to identify Mn- and Cu-MBC, we will test for MBC production in two non-Candida fungal pathogens. Mn-MBC and Cu-MBC from C. albicans will be chemically identified and composition defined by mass spectrometry. Experiments will engage expert collaborators in ENDOR spectroscopy and mass spectrometry. Aim 2: To gain insight into the fundamental biology of MBC. Our preliminary studies show that both Mn- and Cu- MBC can serve as donors of their respective metals for uptake by C. albicans in culture. We shall test the pathway of metal uptake that uses the MBCs and examine metalloenzyme targets for metal delivery by MBC. Time permitting, we shall test the role of Mn- and Cu-MBC in pathogenesis using appropriate C. albicans mutants and a murine model of candidiasis where the fungal pathogen faces metal starvation stress. Overall, successful completion of this 2-year program will reveal the first non-Fe metallophores for the fungal kingdom and new mechanisms by which fungi can capture metal nutrients that are essential for pathogenesis.

包括铜、锰、锌和铁在内的过渡金属是所有微生物病原体的必需营养素。 在获取这些重要元素方面面临挑战，因为宿主认真地试图让微生物挨饿 成功的病原体已经通过统称为营养免疫的过程进化。 逃避营养免疫并从宿主体内捕获金属的复杂方法涉及一种机制； “金属载体”的分泌 金属载体是与细胞外金属结合的小分子（≤1.5 kDa）。 高亲和力并将离子传递给微生物以供吸收。细菌分泌各种铁、铜或金属载体。 锌，但尚未发现致病真菌仅分泌铁金属团。 并且仅在某些物种中白色念珠菌是一种机会性真菌病原体，据信不会产生。 任何种类的金属载体，尽管酵母的发病机制依赖于宿主金属。 挑战了念珠菌属没有金属载体的教条，并获得了白色念珠菌确实存在的证据 事实上产生了对结合 Mn2+ 或 Cu2+ 表现出强选择性的类金属团化合物。 这些 Mn-MBC 和 Cu-MBC 为金属结合络合物 Mn-MBC 和 Cu-MBC 的金属配位位点。 不同，色谱性质也不同，表明 Mn-MBC 和 Cu-MBC 不同 分子暗示，Cu-MBC 的产生，而不是 Mn-MBC，是由 Fe 诱导的，表明 Cu- 的作用。 MBC 在 Fe 稳态中的作用 目前我们还不知道 MBC 分子的化学性质及其性质。 不同真菌之间的发生，或其在真菌发病机制中金属代谢中的作用。 年研究计划，我们将测试念珠菌以外的真菌的 MBC 生产，我们将采用化学方法； 识别白色念珠菌 Mn-MBC 和 Cu-MBC，并将开始探讨其作用机制 目标 1： 调查不同真菌中的 MBC，并使用尺寸对白色念珠菌中的分子进行化学鉴定。 我们开发的排阻色谱/ICP-质谱/ENDOR 光谱方法 为了识别 Mn-MBC 和 Cu-MBC，我们将测试两种非念珠菌真菌病原体中的 MBC 产生情况。 来自白色念珠菌的 Cu-MBC 将通过化学方法进行鉴定，并通过质谱法确定其成分。 实验将邀请 ENDOR 光谱学和质谱学领域的专家合作者参与，目标 2： 深入了解 MBC 的基础生物学 我们的初步研究表明 Mn- 和 Cu-。 MBC 可以作为其各自金属的供体，供培养中的白色念珠菌摄取。 使用 MBC 的金属吸收途径并检查 MBC 进行金属递送的金属酶靶点。 如果时间允许，我们将使用适当的白色念珠菌测试 Mn-和 Cu-MBC 在发病机制中的作用 突变体和念珠菌病小鼠模型，其中真菌病原体面临金属饥饿应激。 这个为期两年的计划的成功完成将揭示真菌王国的第一个非铁金属载体 以及真菌捕获发病机制所必需的金属营养物质的新机制。