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）：口服念珠菌病（由念珠菌引起的OC）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，该抗生素已知会改变微生物菌群。尚未研究念珠菌与口腔微生物组成员之间的相互作用。我们进行了初步研究以：（a）表征口腔细菌微生物组（细菌）和口服真菌微生物组（Mycobiome），以及（b）确定细菌组/霉菌组的变化是否影响念珠菌定植。 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两种真菌。为了确认这种拮抗性相互作用，我们进行了其他研究，并表明：（4）花生培养基（PSM）抑制了念珠菌的生长，粘附，发芽及其形成生物膜的能力，（5）这种抗Candida活性是Pichia的 具体的。 （6）PSM表现出巨大的抗真菌活性，抑制隐孢子，曲霉和镰刀菌。接下来，（7）使用了OC的鼠模型，并表明PSM处理减少了体内的念珠菌感染，如舌头真菌负荷和组织学评估所示，所示。 （8）用蛋白酶-K处理PSM，废除了其抗生物膜活性，而从PSM中提取的代谢产物对念珠菌没有影响，这表明PSM的活性成分/s本质上是蛋白质的。接下来，（9）我们对PSM进行了蛋白质组学分析，并发现了13个具有较高匹配评分的蛋白质（表明较强的身份匹配），包括6种糖酵解酶，3个转录因子，血浆 - 膜膜ATPase，葡萄糖酶，葡萄糖酶和丝氨酸蛋白酶。由于葡萄糖酶和蛋白酶被证明可以介导吡氏对植物真菌的抑制活性，因此假设PSM的抗candida活性是由这些蛋白质介导的。最后，（10）暴露于pepstatin a（蛋白酶抑制剂），废除了与PSM抑制中有关蛋白酶的抗candida活性。在此应用中，我们将纯化并表征葡萄糖酶/蛋白酶蛋白，确定其作用机理，并使用OC的鼠模型验证其功效。当前建议的具体目的是：目标1。纯化和表征PSM的葡萄糖酶和蛋白酶蛋白。 AIM 2。确定紫chi酶/蛋白酶抑制念珠菌的机制。 AIM 3。确定紫葡萄酶/蛋白酶在体内的疗效。了解皮基亚蛋白抑制这些生物的机制对于提供对真菌感染的发病机理和控制的洞察力至关重要。