ROLE OF CANDIDA ACID PROTEINASE IN PATHOGENESIS

念珠菌酸性蛋白酶在发病机制中的作用

• 批准号: 3137336
• 负责人: THOMAS L RAY
• 金额: $ 15.55万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3137336
• 关键词: Candida; antifungal antibody; candidiasis; collagen; complement deficiency; cytotoxicity; disease /disorder model; electron microscopy; enzyme linked immunosorbent assay; enzyme mechanism; enzyme substrate; fungal genetics; gene expression; genetic manipulation; genetic mapping; genetic strain; histology; host organism interaction; immunofluorescence technique; inflammation; keratin; laboratory mouse; laboratory rabbit; laboratory rat; microorganism culture; microorganism immunology; molecular cloning; monoclonal antibody; mutant; opportunistic infections; peptidases; phagocytosis; protease inhibitor; skin infection; virulence

The aim of this proposal is to elucidate the extracellular acid proteinase of Candida spp. (CAP) as a constitutive pathogenic factor in Candida infections by demonstrating: 1) CAP participation in invasion of the skin by Candida in experimental rodent models of cutaneous candidiasis; 2) CAP participation in protecting Candida blastospores from uptake and killing by phagocytic cells; 3) the prevalence of CAP both phenotypically and genomically, in pathogenic and commensal isolates of Candida species, and its correlation with pathogenic and virulent behavior. The role of CAP in cutaneous infections will be performed by studying CAP producing and non-producing isogenic isolates and strains of Candida in experimental rodent models of cutaneous candidiasis. Their ability to invade the epidermis, dermis and fat layers of skin will be assessed by light and electron microscopy for disruption or dissolution of the major proteins of skin, keratin and collagen, which are CAP substrates. CAP inhibition by pepstatin and neutralizing antibody, as well as supplementation with purified CAP, will be studied in these infections. The enzyme will be localized in infected tissue by immunofluorescent microscopy. The role of CAP protecting blastospores from phagocyte uptake and killing will be assessed by standard phagocytosis and killing assays of CAP-producing and non-producing isogenic isolates and species of Candida, in the presence and absence of pepstatin or neutralizing antibody, or supplemental active purified CAP. Direct cytotoxicity of CAP will be assessed by 51Cr-release assays of labelled monocyte, macrophages, and neutrophils. The prevalence of CAP in pathogenic and commensal isolates of Candida will be determined phenotypically by enzyme production in restrictive cultures, and genomically by Southern blot hybridization of restriction endonuclease digests of Candida DNA with a cDNA probe for the CAP encoding gene. Virulent behavior of the isolates will be confirmed in the experimental rodent model of cutaneous candidiasis. This study will characterize the participation of CAP in cutaneous invasion and phagocyte interactions of Candida organisms, demonstrating facilitation of tissue invasion and protection against host inflammatory response. This will be the first study to characterize a virulence factor in Candida species at the genetic level through molecular biology. Data may allow discrimination of commensal and pathogenic isolates for diagnostic testing while revealing critical steps in pathogenesis amenable to therapeutic intervention.

本提案的目的是阐明细胞外酸 念珠菌蛋白酶 (CAP)作为一种组成性致病 念珠菌感染的因素，通过证明：1）CAP 参与念珠菌对皮肤的侵袭 皮肤念珠菌病啮齿动物模型; 2）CAP参与 保护念珠菌芽生孢子不被吸收和杀死， 吞噬细胞; 3）CAP的患病率， 在致病性和念珠菌分离株中， 种，及其与致病性和毒性行为的相关性。 CAP在皮肤感染中的作用将通过以下方式进行： 研究产CAP和不产CAP的同基因分离株， 念珠菌属菌株在实验性啮齿动物模型的皮肤 念珠菌病 它们侵入表皮真皮和脂肪的能力 皮肤层将通过光学和电子显微镜进行评估 用于破坏或溶解皮肤的主要蛋白质、角蛋白 和胶原蛋白，它们是CAP底物。 CAP抑制 胃蛋白酶抑制剂和中和抗体，以及补充 与纯化的CAP，将在这些感染进行研究。 的 酶将通过免疫荧光定位在感染组织中 显微镜 CAP对芽生孢子吞噬细胞摄取的保护作用 通过标准吞噬作用和杀伤作用来评估杀伤作用 产CAP和不产CAP的同基因分离株的测定， 念珠菌属，在存在和不存在胃蛋白酶抑制剂或 中和抗体或补充活性纯化CAP。 CAP的直接细胞毒性将通过51 Cr释放进行评估 标记的单核细胞、巨噬细胞和嗜中性粒细胞的测定。 CAP在致病性和流行性大肠杆菌中的流行情况 念珠菌将通过酶的产生来确定表型 在限制性培养物中，通过Southern印迹在基因组上 念珠菌DNA限制性内切酶杂交 用CAP编码基因的cDNA探针。 恶毒的行为 的分离株将在实验啮齿动物模型中得到证实 皮肤念珠菌病 本研究将描述CAP参与皮肤 念珠菌属生物体的侵袭和吞噬细胞相互作用， 证明了促进组织侵入和保护 抵抗宿主的炎症反应 这将是第一个研究 描述念珠菌属中的毒力因子， 通过分子生物学的方法。 数据可以允许 鉴别细菌和致病性分离物， 诊断测试，同时揭示发病机制的关键步骤 适合于治疗干预。