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。其中一个风险因素是使用抗生素，这是已知的改变微生物菌群。念珠菌与口腔微生物群成员之间的相互作用尚未被调查。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）在hiv感染和未感染的个体中包括14个属，其中13个是常见的；(2)核心口腔真菌组（COM）包括5个属（其中念珠菌和青霉菌在两个队列中共享）；(3)毕赤酵母的死亡丰度与念珠菌丰度的增加一致，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，我们进行了进一步的研究，结果表明：(4)毕赤酵母消耗培养基（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的

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.