Bender Jennifer, Rydzewski Kerstin, Broich Markus, Schunder Eva, Heuner Klaus, Flieger Antje
Division of Bacterial Infections, FG11, Robert Koch-Institut, Burgstrasse 37, Wernigerode 38855, Germany.
J Biol Chem. 2009 Oct 2;284(40):27185-94. doi: 10.1074/jbc.M109.026021. Epub 2009 Jul 29.
Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.
嗜肺军团菌拥有几种能够操纵宿主细胞并造成肺部损伤的磷脂酶。最近，我们发现主要的细胞相关溶血磷脂酶A（PlaB）与已描述的磷脂酶没有同源性，并且在体外细胞内复制过程中并非必需。然而，我们在此表明，PlaB是嗜肺军团菌细胞感染中的主要脂解活性物质，并且PlaB利用典型的丝氨酸-天冬氨酸-组氨酸催化三联体来有效水解磷脂底物。关键残基位于该蛋白的N端一半区域内，嵌入这些活性位点的氨基酸对于PlaB及其同源物而言是独特的。我们进一步表明，PlaB对磷脂酰胆碱而非磷脂酰甘油的催化活性与PlaB的溶血潜力直接相关。尽管PlaB延长的C端功能仍有待阐明，但它对于脂解至关重要，因为去除15个氨基酸就已经消除了酶活性。此外，我们确定PlaB优先水解含有12个或更多碳原子的长链脂肪酸底物。由于磷脂酶作为细菌毒力因子发挥着重要作用，我们检测了嗜肺军团菌临床分离株和非嗜肺军团菌物种中的细胞相关酶活性。所有测试的临床分离株都表现出相当的活性，而在非嗜肺军团菌物种中，只有戈尔曼军团菌和精神军团菌具有与嗜肺军团菌相似的脂解活性并且包含类plaB基因。有趣的是，嗜肺军团菌中的磷脂酰胆碱特异性磷脂酶A活性和溶血潜力更为明显。因此，由PlaB触发的真核细胞膜成分磷脂酰胆碱的水解可能是嗜肺军团菌致病性的一种重要毒力工具。
