Bennett Désirée E, McCreary Christine E, Coleman David C
University of Dublin, The Moyne Institute of Preventive Medicine, Department of Microbiology, Trinity College, Dublin 2, Republic of Ireland.
University of Dublin, School of Dental Science, Department of Oral Medicine and Pathology, Trinity College, Dublin 2, Republic of Ireland.
Microbiology (Reading). 1998 Jan;144 ( Pt 1):55-72. doi: 10.1099/00221287-144-1-55.
Phospholipase C (PLC) enzymes are essential in regulating several important cellular functions in eukaryotes, including yeasts. In this study, PCR was used to identify a gene encoding PLC activity in Candida albicans, using oligonucleotide primers complementary to sequences encoding highly conserved amino acid regions within the X domains of previously characterized eukaryotic phospholipase C genes. The nucleotide sequence of the C. albicans gene, CAPLC1 (2997 bp), was determined from a recombinant clone containing C. albicans 132A genomic DNA; it encoded a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC1) exhibited many of the features common to previously characterized PLCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 18-19 amino acid residues within the X domain and an unusually long N-terminus which did not contain a recognizable EF-hand Ca(2+)-binding domain. An overall amino acid homology of more than 27% with PLCs previously characterized from Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that the CAPLC1 protein is a delta-form of phosphoinositide-specific PLC (PI-PLC). PLC activity was detected in cell-free extracts of both yeast and hyphal forms of C. albicans 132A following 7 h and 24 h growth using the PLC-specific substrate p-nitrophenylphosphorylcholine (p-NPPC). In addition, CAPLC1 mRNA was detected by reverse transcriptase PCR in both yeast and hyphal forms of C. albicans 132A at the same time intervals. Expression of CAPLC1 activity was also detected in extracts of Escherichia coli DH5 alpha harbouring plasmids which contained portions of the CAPLC1 gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses revealed that all C. albicans and Candida dubliniensis isolates examined possessed sequences homologous to CAPLC1. Sequences related to CAPLC1 were detected in some but not all isolates of Candida tropicalis, Candida glabrata and Candida parapsilosis tested, but not in the isolates of Candida krusei, Candida kefyr, Candida guillermondii and Candida lusitaniae examined. This paper reports the first description of the cloning and sequencing of a PLC gene from a pathogenic yeast species.
磷脂酶C（PLC）酶在调节真核生物（包括酵母）的几种重要细胞功能中起着至关重要的作用。在本研究中，使用聚合酶链反应（PCR）来鉴定白色念珠菌中编码PLC活性的基因，所用的寡核苷酸引物与先前鉴定的真核磷脂酶C基因X结构域内编码高度保守氨基酸区域的序列互补。从含有白色念珠菌132A基因组DNA的重组克隆中确定了白色念珠菌基因CAPLC1（2997 bp）的核苷酸序列；它编码一个由1099个氨基酸组成的多肽，预测分子量为124.6 kDa。该多肽（CAPLC1）推导的氨基酸序列表现出许多先前鉴定的PLC共有的特征，包括特定的X和Y催化结构域。CAPLC1蛋白还表现出几个独特的特征，包括X结构域内一段新的18 - 19个氨基酸残基的延伸以及一个异常长的N末端，该末端不包含可识别的EF手型Ca(2+)结合结构域。与先前从酿酒酵母和粟酒裂殖酵母鉴定的PLC的总体氨基酸同源性超过27%，这表明CAPLC1蛋白是磷酸肌醇特异性PLC（PI - PLC）的δ型。使用PLC特异性底物对硝基苯基磷酰胆碱（p - NPPC），在白色念珠菌132A的酵母和菌丝体形式的无细胞提取物中，分别在生长7小时和24小时后检测到了PLC活性。此外，通过逆转录酶PCR在相同时间间隔的白色念珠菌132A的酵母和菌丝体形式中均检测到了CAPLC1 mRNA。在含有缺失编码部分N末端序列的CAPLC1基因片段的质粒的大肠杆菌DH5α提取物中也检测到了CAPLC1活性的表达。Southern杂交和PCR分析表明，所有检测的白色念珠菌和都柏林念珠菌分离株都具有与CAPLC1同源的序列。在一些但并非所有测试的热带念珠菌、光滑念珠菌和近平滑念珠菌分离株中检测到了与CAPLC1相关的序列，但在所检测的克鲁斯念珠菌、凯弗念珠菌、季也蒙念珠菌和葡萄牙念珠菌分离株中未检测到。本文首次报道了致病酵母物种中PLC基因的克隆和测序。
