Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA.
Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
mBio. 2021 Feb 9;12(1):e03458-20. doi: 10.1128/mBio.03458-20.
Diversion of the -containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires' pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in have been shown to interfere with various host pathways and processes, but no effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of in both hMDMs and amoeba. The substitution defects of P and D are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of is indispensable for phagosome biogenesis and lysosomal evasion. Intracellular proliferation of within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires' pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the -containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway. The pathogen has evolved to inject ∼350 protein effectors into the host cell where they modulate various host processes, but no effector has ever been identified to be indispensable for lysosomal evasion. We show that the MavE effector localizes to the poles of the LCV membrane and is essential for lysosomal evasion and intracellular growth of and for intrapulmonary proliferation in mice. The crystal structure of MavE shows an NPxY eukaryotic motif essential for ER-mediated remodeling and lysosomal evasion by the LCV. Therefore, the MavE effector of is indispensable for phagosome biogenesis and lysosomal evasion.
含空泡的液泡（LCV）从宿主内体溶酶体降解途径的转移是军团菌肺炎表现所必需的主要毒力特征之一。在 中鉴定的约 350 个 Dot/Icm 注射效应物中的许多已被证明干扰各种宿主途径和过程，但从未鉴定出任何 效应物对于溶酶体逃避是必不可少的。虽然大多数 的单个效应物突变体在巨噬细胞中没有表现出缺陷表型，但我们表明 MavE 效应物对于人类单核细胞衍生的巨噬细胞（hMDM）和变形虫中的 和在小鼠中的肺内增殖是必需的。 的缺失突变体不能用内质网（ER）衍生的囊泡重塑 LCV，并且被运送到溶酶体，在那里它被降解，类似于福尔马林杀死的细菌。在 hMDM 的感染过程中，MavE 效应物定位于 LCV 膜的两极。以 1.8Å 的分辨率解析的 MavE 晶体结构揭示了一个 C 末端跨膜螺旋、三个酪氨酸基分拣基序拷贝和一个 NPxY 真核基序，该基序结合存在于信号传导和衔接真核蛋白上的磷酸酪氨酸结合结构域。NPxY 基序内的两个点突变导致在 hMDM 和变形虫中 的衰减。P 和 D 的取代缺陷与携带突变体的空泡不能被 ER 重塑以及导致空泡与溶酶体融合从而导致细菌降解有关。因此， 的 MavE 效应物对于吞噬体发生和溶酶体逃避是必不可少的。在人类肺泡巨噬细胞中的空泡内 的细胞内增殖对于军团病的表现是必不可少的。病原体在囊泡内的生长完全依赖于 ER 重塑含 的空泡（LCV）及其逃避内体溶酶体降解途径。病原体已经进化到将约 350 种蛋白质效应物注入宿主细胞，在那里它们调节各种宿主过程，但从未鉴定出任何 效应物对于溶酶体逃避是必不可少的。我们表明，MavE 效应物定位于 LCV 膜的两极，对于溶酶体逃避和 的细胞内生长以及在小鼠中的肺内增殖是必需的。MavE 的晶体结构显示 NPxY 真核基序对于 ER 介导的重塑和 LCV 的溶酶体逃避是必不可少的。因此， 的 MavE 效应物对于吞噬体发生和溶酶体逃避是必不可少的。
