Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois, USA.
Department of Chemistry and Biochemistry, Queen Mary University of London, London, United Kingdom.
mBio. 2018 Apr 17;9(2):e00528-18. doi: 10.1128/mBio.00528-18.
genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of , although these genes had been considered dispensable on the basis of the behavior of mutants lacking either and or A mutant showed even higher levels of and transcripts, and a mutant showed heightening of 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 mutants have an ~50-fold defect during infection of 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 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 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 genus. LapA was notable for having as its closest homologue an protein. Transmission of to humans is facilitated by its ability to grow in species. We previously documented that type II secretion (T2S) promotes infection of 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 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 genus in unique ways. The results of this study represent a major advance in understanding ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.
基因编码 LapA、LapB 和 PlaC 被鉴定为在感染过程中高度上调的 II 型分泌 (T2S) 基因，尽管这些基因在缺乏任何 和 或 突变体的行为基础上被认为是可有可无的，突变体显示出更高水平的 和 转录物，而 突变体显示出 mRNA 水平的升高，表明 LapA/B 氨肽酶的作用与 PlaC 酰基转移酶的作用是互补的。因此，我们构建了双突变体，并发现 突变体在感染过程中存在约 50 倍的缺陷。这些数据首次揭示了 LapA 在任何感染中的重要性；因此，我们纯化了 LapA 并定义了它的晶体结构、由另一种 T2S 依赖性蛋白酶 (ProA) 激活以及广泛的底物特异性。当在变形虫感染培养基中补充氨基酸时， 突变体的缺陷得到了逆转，这意味着 LapA 产生氨基酸用于营养。由于 LapA 和 PlaC 数据并未完全解释 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型分泌蛋白 (NttD)，它促进了 感染，突变体显示出更大的（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 和 属内和属外具有不同的分布模式。LapA 的特点是其最接近的同源物是 蛋白。通过其在 种中的生长能力，促进了对人类的传播。我们之前记录了 II 型分泌 (T2S) 促进了 对 的感染。利用转录分析和蛋白质组学、双突变体和三突变体以及晶体结构，我们定义了三个分泌底物/效应物，它们在很大程度上阐明了 T2S 在 感染过程中的作用。特别有趣的是酰基转移酶 (PlaC) 和氨肽酶 (LapA) 之间的独特功能重叠、LapA 的广泛底物特异性和真核蛋白样特征以及 NttD 的新颖性。将 LapA 与氨基酸获取联系起来，我们首次定义了分泌氨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，揭示了效应物源自不同的来源，并以独特的方式分布在 属内。这项研究的结果代表了对 生态和发病机制、细菌分泌以及细胞内寄生进化的理解的重大进展。
