Mechanism of antifungal action of Pichia proteins

描述(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，包括感染艾滋病毒、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，众所周知，抗生素会改变微生物菌群。念珠菌与口腔微生物组成员之间的相互作用尚未被研究。我们进行了初步研究，以：(A)确定口腔细菌微生物组(细菌组)和口腔真菌微生物组(真菌生物组)的特征，以及(B)确定细菌组/真菌生物组的变化是否影响念珠菌的定植。结果表明：(1)HIV感染者和非感染者的核心口腔菌群(COB)由14个属组成，其中13个属是常见的；(2)核心口腔真菌群(COM)由5个属组成(其中念珠菌和青霉菌在两组人群中共享)；(3)酵母毕赤酵母的死亡丰度与念珠菌丰度的增加相吻合，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，我们进行了进一步的研究，结果表明：(4)毕赤酵母废液(PSM)抑制了念珠菌的生长、黏附、萌发及其形成生物膜的能力，(5)这种抗念珠菌的活性就是毕赤酵母 具体的。(6)PSM具有广泛的抗真菌活性，对隐球菌、曲霉和镰刀菌也有抑制作用。接下来，(7)使用了一种小鼠OC模型，结果表明，PSM治疗可以减少体内念珠菌的感染，如减少舌部真菌负荷和组织学评估。(8)PSM经蛋白酶-K处理后，其抗生物被膜活性减弱，而PSM的代谢产物对念珠菌无影响，表明PSM的活性成分/S为蛋白质类物质。接下来，(9)我们对PSM进行了蛋白质组学分析，发现了13个匹配分数高的蛋白质，包括6个糖酵解酶、3个转录因子、1个质膜ATPase、1个葡聚糖酶和1个丝氨酸蛋白酶。由于葡聚糖酶和蛋白水解酶参与了毕赤酵母对植物真菌的抑制作用，因此我们推测PSM的抗念珠菌活性是通过这两种蛋白来实现的。最后，(10)胃抑素A(蛋白酶抑制剂)抑制PSM的抗念珠菌活性，提示PSM抑制PSM的蛋白水解酶活性。在这项应用中，我们将纯化和鉴定葡聚糖酶/蛋白酶蛋白，确定它们的作用机制/S，并用小鼠OC模型验证它们的有效性。本方案的具体目的是：目的1.分离纯化PSM的葡聚糖酶和蛋白酶蛋白，并对其进行性质鉴定。目的2.确定毕赤酵母葡聚糖酶/蛋白酶抑制念珠菌的机制/S。目的3.测定毕赤酵母葡聚糖酶/蛋白水解酶的体内活性。了解毕赤酵母蛋白抑制这些微生物的机制/S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.