Kutty Geetha, Shroff Robert, Kovacs Joseph A
Critical Care Medicine Department, NIH Clinical Center, NIH, Bethesda, Maryland, USA.
Eukaryot Cell. 2013 Oct;12(10):1349-55. doi: 10.1128/EC.00122-13. Epub 2013 Jul 26.
Major surface glycoprotein (Msg), the most abundant cell surface protein of Pneumocystis, plays an important role in the interaction of this opportunistic pathogen with host cells, and its potential for antigenic variation may facilitate evasion of host immune responses. In the present study, we have identified and characterized the promoter region of msg in 3 species of Pneumocystis: P. carinii, P. jirovecii, and P. murina. Because Pneumocystis cannot be cultured, promoter activity was measured in Saccharomyces cerevisiae, a related fungus, using a yeast vector modified to utilize the gene coding for Renilla luciferase as a reporter gene. The 5'-flanking sequences of msg from all three Pneumocystis species showed considerable promoter activity, with increases in luciferase activity up to 15- to 44-fold above baseline. Progressive deletions helped define an ∼13-bp sequence in each Pneumocystis species that appears to be critical for promoter activity. Electrophoretic mobility shift analysis using P. carinii-specific msg promoter sequences demonstrated binding of nuclear proteins of S. cerevisiae. The 144-bp 5'-flanking region of P. murina msg showed 72% identity to that of P. carinii. The 5'-flanking region of P. jirovecii msg showed 58 and 61% identity to those of P. murina and P. carinii, respectively. The msg promoter is a good candidate for inclusion in a construct designed for genetic manipulation of Pneumocystis species.
主要表面糖蛋白（Msg）是肺孢子菌中最丰富的细胞表面蛋白，在这种机会性病原体与宿主细胞的相互作用中发挥重要作用，其抗原变异的可能性可能有助于逃避宿主免疫反应。在本研究中，我们鉴定并表征了三种肺孢子菌（卡氏肺孢子菌、耶氏肺孢子菌和鼠肺孢子菌）中msg的启动子区域。由于肺孢子菌无法培养，因此使用经过修饰以利用海肾荧光素酶编码基因作为报告基因的酵母载体，在相关真菌酿酒酵母中测量启动子活性。来自所有三种肺孢子菌的msg的5'侧翼序列均显示出相当的启动子活性，荧光素酶活性比基线增加了15至44倍。逐步缺失有助于确定每种肺孢子菌中一个约13bp的序列，该序列似乎对启动子活性至关重要。使用卡氏肺孢子菌特异性msg启动子序列进行的电泳迁移率变动分析表明酿酒酵母核蛋白与之结合。鼠肺孢子菌msg的144bp 5'侧翼区域与卡氏肺孢子菌的该区域具有72%的同一性。耶氏肺孢子菌msg的5'侧翼区域与鼠肺孢子菌和卡氏肺孢子菌的该区域分别具有58%和61%的同一性。msg启动子是用于肺孢子菌遗传操作的构建体中的一个很好的候选者。
