Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas.
Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas.
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DOI：
10.1128/mbio.00528-18
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发表时间：
2018-04-17
期刊：
影响因子：
6.4
通讯作者：
Cianciotto NP
中科院分区：
文献类型：
--
作者：
White RC;Gunderson FF;Tyson JY;Richardson KH;Portlock TJ;Garnett JA;Cianciotto NP
嗜肺军团菌编码LapA、LapB和plac的基因被鉴定为在感染卡氏棘阿米巴过程中表达最高的II型分泌(T2S)基因，尽管根据缺乏LapA、LapB和plac的突变株的行为，这些基因被认为是可有可无的。一个plac突变体表现出更高的LapA和LapB转录本水平，而一个Lapa LapB突变体表现出plac mRNA水平的升高，这表明LapA/B氨基肽酶的作用相对于plac酰基转移酶的作用是代偿的。因此，我们制作了双突变体，发现Lapa plac突变体在感染卡氏不动杆菌过程中存在~50倍的缺陷。这些数据第一次揭示了LapA在任何类型的感染中的重要性；因此，我们纯化了LapA并确定了它的晶体结构，被另一种T2S依赖的蛋白酶(ProA)激活，以及广泛的底物特异性。当阿米巴感染培养液中添加氨基酸时，Lapa plac突变体的缺陷被逆转，这意味着LapA产生了营养所需的氨基酸。由于LAPA和PLAC的数据不能完全解释T2S在感染中的作用，我们通过蛋白质组学分析鉴定了一种新的促进卡氏不动杆菌感染的分泌蛋白(NttD)。一个LapA Plac nttD突变体表现出更大的(100倍)缺陷，表明LapA、Plac和NttD数据在很大程度上解释了T2S的重要性。LapA、Plac和NttD样蛋白在军团菌属内外有不同的分布模式。值得注意的是，Lapa拥有一种与其最接近的同源物--卡氏锥虫蛋白。嗜肺性乳杆菌通过在棘阿米巴物种中生长的能力促进了向人类的传播。我们之前曾记录过，II型分泌物(T2S)促进卡氏不动杆菌的嗜肺乳杆菌感染。利用转录分析和蛋白质组学、双突变体和三重突变体以及晶体结构，我们定义了三个分泌底物/效应子，在很大程度上阐明了T2S在感染卡氏不动杆菌过程中的作用。特别有趣的是酰基转移酶(PLAC)和氨基肽酶(LAPA)之间独特的功能重叠，LAPA广泛的底物专一性和类真核蛋白质的特性，以及NttD的新颖性。将LAPA与氨基酸的获取联系起来，我们第一次确定了分泌型氨基肽酶在细胞内感染中的重要性。生物信息学研究，以前没有应用于T2S，揭示了效应器来自不同的来源，并以独特的方式分布在军团菌属内。这项研究的结果在了解军团菌的生态和发病机制、细菌分泌和细胞内寄生的进化方面取得了重大进展。
Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC. A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii. These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii. A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein. Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii. Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii. Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.