Mechanism of antifungal action of Pichia proteins

毕赤酵母蛋白的抗真菌作用机制

• 批准号: 9422694
• 负责人: Mahmoud A Ghannoum
• 金额: $ 39.63万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-14 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9422694
• 关键词: ATP phosphohydrolase; Adherence; Affect; Affinity Chromatography; Age; Ammonium Sulfate; Antibiotics; Antibodies; Antifungal Agents; Apoptosis; Aspergillus; Biological Assay; Candida; Cell membrane; Chromatography; Clinical; Complication; Cryptococcus; Data; Diabetes Mellitus; Disease; Dose; Endopeptidase K; Enzymes; Ethnic Origin; Evaluation; Exhibits; Exposure to; Extravasation; Fluconazole; Fractionation; Fusarium; Germination; Glucans; Growth; HIV; Histologic; Immunocompromised Host; In Vitro; Individual; Infant; Infection; Ion Exchange; Lectin; Malignant Neoplasms; Mediating; Methods; Microbial Biofilms; Morphology; Mycoses; Nature; Nystatin; Oral; Oral Characters; Oral candidiasis; Organism; Oxidative Stress; Participant; Pathogenesis; Pathogenicity; Patients; Penicillium; Pepstatins; Peptide Hydrolases; Pichia; Prevention; Protease Inhibitor; Proteins; Proteomics; Risk Factors; Sepharose; Serine Protease; Substrate Specificity; Temperature; Tissues; Tongue; Topical application; Transcription Factor 3; Western Blotting; Yeasts; aspergillopepsin II; bacteriome; cohort; efficacy testing; fungus; in vivo; inhibitor/antagonist; insight; liquid chromatography mass spectrometry; member; microbiota; mouse model; mycobiome; novel; oral bacteria; oral care; oral fungal; oral microbiome; plant fungi; proteinase In; public health relevance; sex; standard of care; treatment duration

DESCRIPTION (provided by applicant): Oral candidiasis (OC, caused by Candida) is a major complication in immunosuppressed patients, including those infected with HIV, cancer, and diabetes, as well as infants. One of the risk factors for OC is the use of antibiotics, which is known to alter the microbial flora. Interactions between Candida and members of the oral microbiome have not been investigated. We performed preliminary studies to: (a) characterize the oral bacterial microbiome (bacteriome) and oral fungal microbiome (mycobiome), and (b) determine whether changes in bacteriome/mycobiome affect Candida colonization. We showed: (1) core oral bacteriome (COB) comprises 14 genera in HIV-infected and uninfected individuals, of which 13 were common, (2) core oral mycobiome (COM) comprised 5 genera (of which Candida and Penicillium were shared between the two cohorts), (3) deceased abundance of the yeast Pichia coincided with increase in Candida abundance, suggesting an antagonistic association between these two fungi. To confirm this antagonistic interaction, we performed additional studies and showed that: (4) Pichia spent media (PSM) inhibited Candida growth, adherence, germination, and its ability to form biofilms, (5) this anti-Candida activity was Pichia specific. (6) PSM exhibited broad antifungal activity inhibiting Cryptococcus, Aspergillus and Fusarium as well. Next, (7) used a murine model of OC and showed that PSM-treatment reduced Candida infection in vivo, as shown by reduction in tongue fungal load and histological evaluation. (8) Treatment of PSM with proteinase-K abrogated its anti-biofilm activity, while metabolites extracted from PSM had no effect on Candida, showing that the active component/s of PSM is proteinaceous in nature. Next, (9) we performed proteomics analysis of PSM and found 13 proteins with a high match score (indicating strong identity match), including 6 glycolytic enzymes, 3 transcription factors, a plasma-membrane ATPase, a glucanase, and a serine proteinase. Since, glucanase and proteinase were shown to mediate the inhibitory activity of Pichia against plant fungi we hypothesize that the anti-Candida activity of PSM is mediated by these proteins. Finally, (10) exposure to pepstatin A (proteinase inhibitor) abrogated the anti-Candida activity of PSM implicating proteinase in PSM inhibition. In this application, we will purify and characterize the glucanase/proteinase proteins, determine their mechanism/s of action, and validate their efficacy using a murine model of OC. The specific aims of the current proposal are: Aim 1. Purify and characterize glucanase and proteinase proteins of PSM. Aim 2. Determine the mechanism/s by which Pichia glucanase/proteinase inhibit Candida. Aim 3. Determine the efficacy of Pichia glucanase/proteinase in vivo. Understanding the mechanism/s by which Pichia proteins inhibit these organisms is critical in providing insight into the pathogenesis and control of fungal infections.

描述（由申请人提供）：口腔念珠菌病（OC，由念珠菌引起）是免疫抑制患者的主要并发症，包括感染 HIV、癌症和糖尿病的患者以及婴儿。 OC 的危险因素之一是使用抗生素，众所周知，抗生素会改变微生物菌群。念珠菌和口腔微生物组成员之间的相互作用尚未得到研究。我们进行了初步研究：（a）表征口腔细菌微生物组（细菌组）和口腔真菌微生物组（真菌组），以及（b）确定细菌组/真菌组的变化是否影响念珠菌定植。我们发现：（1）核心口腔细菌组（COB）由 HIV 感染者和未感染者的 14 个属组成，其中 13 个属常见，（2）核心口腔真菌组（COM）由 5 个属组成（其中念珠菌属和青霉菌属为两个群体共有），（3）酵母毕赤酵母丰度的减少与念珠菌丰度的增加相一致，表明这些属之间存在拮抗关系 两种真菌。为了证实这种拮抗相互作用，我们进行了额外的研究并表明：（4）毕赤酵母废培养基（PSM）抑制念珠菌生长、粘附、发芽及其形成生物膜的能力，（5）这种抗念珠菌活性是毕赤酵母 具体的。 (6) PSM 表现出广泛的抗真菌活性，还能抑制隐球菌、曲霉和镰刀菌。接下来，(7) 使用 OC 小鼠模型，结果表明，PSM 治疗可减少体内念珠菌感染，如舌头真菌负荷的减少和组织学评估所示。 (8)用蛋白酶-K处理PSM消除了其抗生物膜活性，而从PSM中提取的代谢物对念珠菌没有影响，表明PSM的活性成分本质上是蛋白质。接下来，（9）我们对PSM进行了蛋白质组学分析，发现了13个具有高匹配分数（表明强同一性匹配）的蛋白质，包括6种糖酵解酶、3种转录因子、一种质膜ATP酶、一种葡聚糖酶和一种丝氨酸蛋白酶。由于葡聚糖酶和蛋白酶被证明介导毕赤酵母对植物真菌的抑制活性，我们假设 PSM 的抗念珠菌活性是由这些蛋白质介导的。最后，(10) 暴露于胃酶抑素 A（蛋白酶抑制剂）会消除 PSM 的抗念珠菌活性，这表明蛋白酶在 PSM 抑制中发挥作用。在此应用中，我们将纯化和表征葡聚糖酶/蛋白酶蛋白，确定其作用机制，并使用 OC 鼠模型验证其功效。当前提案的具体目标是： 目标 1. 纯化并表征 PSM 的葡聚糖酶和蛋白酶蛋白。目标 2. 确定毕赤酵母葡聚糖酶/蛋白酶抑制念珠菌的机制。目标 3. 确定毕赤酵母葡聚糖酶/蛋白酶的体内功效。了解毕赤酵母蛋白抑制这些生物体的机制对于深入了解真菌感染的发病机制和控制至关重要。