Grigore Daniela, Meade John C
Department of Microbiology, University of Mississippi Medical Center, Jackson, 39216-4505, USA.
J Eukaryot Microbiol. 2006 May-Jun;53(3):157-64. doi: 10.1111/j.1550-7408.2006.00089.x.
The opportunistic fungus Pneumocystis is the etiologic agent of an interstitial plasma cell pneumonia that primarily afflicts immunocompromised individuals. Like other fungi Pneumocystis maintains a H(+) plasma membrane gradient to drive nutrient uptake and regulates intracellular pH by ATP-dependent proton efflux. Previously, we identified a Pneumocystis gene, PCA1, whose predicted protein product was homologous to fungal proton pumps. In this study, we show by functional complementation in a Saccharomyces strain whose endogenous PMA1 proton pump activity is repressed that the Pneumocystis PCA1 encodes a H(+)-ATPase. The properties of PCA1 characterized in this system closely resemble those of yeast PMA1. Yeast expressing PCA1 grow at low pH and are able to acidify the external media. Maximal enzyme activity (V(max)) and efficiency of substrate utilization (K(m)) in plasma membranes were nearly identical for PCA1 and PMA1. PCA1 contains an inhibitory COOH-terminal domain; removal of the final 40 amino acids significantly increased V(max) and growth at pH 6.5. PCA1 activity was inhibited by proton pump inhibitors omeprazole and lansoprazole, but was unaffected by H(+)/K(+)-ATPase inhibitor SCH28080. Thus, H(+) homeostasis in Pneumocystis is likely regulated as in other fungi. This work also establishes a system for screening PCA1 inhibitors to identify new anti-Pneumocystis agents.
机会性真菌肺孢子菌是一种间质性浆细胞肺炎的病原体，主要感染免疫功能低下的个体。与其他真菌一样，肺孢子菌维持质膜H(+)梯度以驱动营养物质摄取，并通过ATP依赖的质子外流调节细胞内pH值。此前，我们鉴定了一个肺孢子菌基因PCA1，其预测的蛋白质产物与真菌质子泵同源。在本研究中，我们通过在一种内源性PMA1质子泵活性受到抑制的酿酒酵母菌株中进行功能互补实验表明，肺孢子菌PCA1编码一种H(+)-ATP酶。在该系统中表征的PCA1的特性与酵母PMA1的特性非常相似。表达PCA1的酵母能在低pH条件下生长，并能够酸化外部培养基。PCA1和PMA1在质膜中的最大酶活性（V(max)）和底物利用效率（K(m)）几乎相同。PCA1含有一个抑制性的COOH末端结构域；去除最后40个氨基酸显著提高了V(max)以及在pH 6.5时的生长能力。PCA1的活性受到质子泵抑制剂奥美拉唑和兰索拉唑的抑制，但不受H(+)/K(+)-ATP酶抑制剂SCH28080的影响。因此，肺孢子菌中的H(+)稳态可能与其他真菌一样受到调节。这项工作还建立了一个筛选PCA1抑制剂以鉴定新型抗肺孢子菌药物的系统。
