Ghannoum M A
Center for Medical Mycology, Mycology Reference Laboratory, Case Western Reserve University, Cleveland, Ohio, USA.
Nihon Ishinkin Gakkai Zasshi. 1998;39(2):55-9. doi: 10.3314/jjmm.39.55.
Microbial pathogens use a number of genetic strategies to invade the host and cause infection. These common themes are found throughout microbial virulence factors. Secretion of enzymes, such as phospholipase, has been proposed as one of these themes which is used by bacteria, parasite, and pathogenic fungi. The role of extracellular phospholipase as a potential virulence factor in pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus has gained credence recently. In this address data implicating phospholipase as a virulence factor in Cryptococcus neoformans and Aspergillus fumigatus will be presented. This will be followed by a more detailed description of our molecular and biochemical approaches we used to more definitively delineate the role of phospholipase in the virulence of C. albicans. First, we purified the phospholipase B protein, the dominant phospholipase secreted by C. albicans, obtained the amino acid sequence of its N-terminus and an internal peptide fragment, and used this information to clone the gene encoding the protein using a PCR-based approach. Nucleotide sequence analysis revealed an ORF of 1818 bp that predicted for a pre-protein of 605 amino acid residues. The deduced amino acid sequences of the cloned gene (PLB 1) showed 42.3%, 45%, and 47.8% overall sequence identity, with the reported sequences of phospholipase B cloned from Penicillium notatum, Saccharomyces cerevisiae, and Saccharomyces rosei, respectively. Second, using targeted gene disruption, URA blaster, we created C. albicans null mutants which failed to secrete phospholipase B. Third, we tested the ability of these isogenic strain pairs to cause lethality using a murine model of hematogenously disseminated candidiasis. Our data demonstrate that the parent phospholipase-producing strain caused more fatality in mice, while the null phospholipase-deficient strain was avirulent. Importantly, the parent and null mutants had similar growth and germination rates. These data prove that phospholipase B is essential for candidal virulence, and pave the way for studies directed at determining the mechanism/s through which phospholipase modulate candidal virulence. Understanding phospholipase as a common theme in fungal pathogenicity is critical for developing new antifungal strategies based on anti-virulence.
微生物病原体利用多种遗传策略侵入宿主并引发感染。这些共同特点在微生物毒力因子中普遍存在。诸如磷脂酶等酶的分泌已被认为是细菌、寄生虫和致病真菌所采用的此类特点之一。细胞外磷脂酶作为包括白色念珠菌、新型隐球菌和曲霉菌在内的致病真菌中一种潜在毒力因子的作用，最近已得到认可。在本次演讲中，将展示有关磷脂酶作为新型隐球菌和烟曲霉中毒力因子的数据。接下来将更详细地描述我们用于更明确界定磷脂酶在白色念珠菌毒力中作用的分子和生化方法。首先，我们纯化了白色念珠菌分泌的主要磷脂酶——磷脂酶B蛋白，获得了其N端和一个内部肽段的氨基酸序列，并利用这些信息通过基于PCR的方法克隆了编码该蛋白的基因。核苷酸序列分析揭示了一个1818 bp的开放阅读框，预测其前体蛋白有605个氨基酸残基。克隆基因（PLB1）推导的氨基酸序列与分别从点青霉、酿酒酵母和玫瑰酵母克隆的磷脂酶B的报道序列总体序列同一性分别为42.3%、45%和47.8%。其次，我们使用靶向基因敲除技术“URA blaster”创建了不分泌磷脂酶B的白色念珠菌缺失突变体。第三，我们使用血行播散性念珠菌病小鼠模型测试了这些同基因菌株对的致死能力。我们的数据表明，产生磷脂酶的亲本菌株在小鼠中导致更多死亡，而缺乏磷脂酶的缺失菌株无毒力。重要的是，亲本菌株和缺失突变体具有相似的生长和萌发率。这些数据证明磷脂酶B对念珠菌的毒力至关重要，并为旨在确定磷脂酶调节念珠菌毒力的机制的研究铺平了道路。将磷脂酶理解为真菌致病性的一个共同特点对于开发基于抗毒力的新抗真菌策略至关重要。
