UCIBIO- Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, NOVA University Lisbon, Caparica, Portugal.
Núcleo de Bioinformática, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal.
Front Cell Infect Microbiol. 2022 May 31;12:864626. doi: 10.3389/fcimb.2022.864626. eCollection 2022.
is an accidental human pathogen that causes the potentially fatal Legionnaires' disease, a severe type of pneumonia. The main virulence mechanism of is a Type 4B Secretion System (T4SS) named Icm/Dot that transports effector proteins into the host cell cytosol. The concerted action of effectors on several host cell processes leads to the formation of an intracellular Legionella-containing vacuole that is replication competent and avoids phagolysosomal degradation. To date over 300 Icm/Dot substrates have been identified. In this study, we searched the genome of a strain (Pt/VFX2014) responsible for the second largest outbreak worldwide (in Vila Franca de Xira, Portugal, in 2014) for genes encoding potential novel Icm/Dot substrates. This strain Pt/VFX2014 belongs to serogroup 1 but phylogenetically segregates from all other serogroup 1 strains previously sequenced, displaying a unique mosaic genetic backbone. The ability of the selected putative effectors to be delivered into host cells by the T4SS was confirmed using the TEM-1 β-lactamase reporter assay. Two previously unknown Icm/Dot effectors were identified, VFX05045 and VFX10045, whose homologs Lpp1450 and Lpp3070 in clinical strain Paris were also confirmed as T4SS substrates. After delivery into the host cell cytosol, homologs VFX05045/Lpp1450 remained diffused in the cell, similarly to Lpp3070. In contrast, VFX10045 localized to the host cell nucleus. To understand how VFX10045 and Lpp3070 (94% of identity at amino acid level) are directed to distinct sites, we carried out a comprehensive site-directed mutagenesis followed by analyses of the subcellular localization of the mutant proteins. This led to the delineation of region in the C-terminal part (residues 380 to 534) of the 583 amino acid-long VFX10045 as necessary and sufficient for nuclear targeting and highlighted the fundamental function of the VFX10045-specific R440 and I441 residues in this process. These studies revealed a strain-specific nucleotropism for new effector VFX10045/Lpp3070, which anticipates distinct functions between these homologs.
是一种偶然的人类病原体，可引起潜在致命的军团病，这是一种严重的肺炎。的主要毒力机制是一种名为 Icm/Dot 的 4B 型分泌系统（T4SS），它将效应蛋白输送到宿主细胞质中。效应蛋白在几个宿主细胞过程中的协同作用导致形成一个含有军团菌的、能够复制的、逃避吞噬体降解的细胞内空泡。迄今为止，已经鉴定出超过 300 种 Icm/Dot 底物。在这项研究中，我们在负责全球第二大 爆发的 菌株（Pt/VFX2014）的基因组中搜索了编码潜在新型 Icm/Dot 底物的基因。该菌株 Pt/VFX2014 属于血清群 1，但在系统发育上与以前测序的所有其他血清群 1 菌株分离，显示出独特的镶嵌遗传主干。使用 TEM-1 β-内酰胺酶报告测定法证实了所选推定效应物通过 T4SS 递送入宿主细胞的能力。鉴定出两种以前未知的 Icm/Dot 效应物，VFX05045 和 VFX10045，其在临床株 Paris 中的同源物 Lpp1450 和 Lpp3070 也被确认为 T4SS 底物。在递送入宿主细胞质后，同源物 VFX05045/Lpp1450 像 Lpp3070 一样在细胞中扩散。相比之下，VFX10045 定位于宿主细胞核。为了了解 VFX10045 和 Lpp3070（氨基酸水平上 94%的同一性）如何被导向不同的位点，我们进行了全面的定点突变，然后分析突变蛋白的亚细胞定位。这导致了在 583 个氨基酸长的 VFX10045 的 C 末端部分（残基 380 至 534）中划定了一个区域，该区域对于核靶向是必要和充分的，并突出了 VFX10045 特异性 R440 和 I441 残基在该过程中的基本功能。这些研究揭示了新效应物 VFX10045/Lpp3070 的菌株特异性核嗜性，并预测了这些同源物之间的不同功能。
