Mechanism of antifungal action of Pichia proteins

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的风险因素之一是使用抗生素，已知抗生素会改变微生物植物群。念珠菌和口腔微生物组成员之间的相互作用尚未研究。我们进行了初步研究以：（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殖。我们展示了：（1）核心口腔细菌组（COB）由14个属组成，其中13个属是常见的;（2）核心口腔真菌组（COM）由5个属组成，其中念珠菌属和青霉属是两个群体共有的;（3）酵母毕赤酵母菌的丰度下降与念珠菌属丰度增加相一致，表明这两种真菌之间存在拮抗关系。为了证实这种拮抗作用，我们进行了额外的研究，结果表明：（4）毕赤酵母菌废培养基（PSM）抑制念珠菌的生长，粘附，萌发及其形成生物膜的能力，（5）这种抗念珠菌活性是毕赤酵母菌 特定. (6)PSM对隐球菌属、曲霉属和镰刀菌属均有较好的抑菌活性。接下来，（7）使用OC的鼠模型，并显示PSM治疗减少了体内念珠菌感染，如舌真菌负荷减少和组织学评价所示。(8)用蛋白酶-K处理PSM废除了其抗生物膜活性，而从PSM提取的代谢产物对念珠菌没有影响，表明PSM的活性组分本质上是蛋白质。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（指示强同一性匹配）的蛋白质，包括6个糖酵解酶、3个转录因子、质膜ATP酶、葡聚糖酶和丝氨酸蛋白酶。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，我们推测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）消除了PSM的抗念珠菌活性，表明蛋白酶参与了PSM抑制。在本申请中，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小鼠模型验证其功效。本提案的具体目标是：目标1。对PSM的葡聚糖酶和蛋白酶蛋白进行了纯化和鉴定。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/蛋白酶在体内的功效。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。

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.