Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector.
Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector.
复制标题
DOI：
10.1371/journal.ppat.1000822
复制
发表时间：
2010-03-19
期刊：
影响因子：
6.7
通讯作者：
Luo ZQ
中科院分区：
文献类型：
--
作者：
Xu L;Shen X;Bryan A;Banga S;Swanson MS;Luo ZQ
嗜肺军团菌是引起军团病的细胞内病原体。这种细菌使用Dot/Icm IV型分泌系统将大量细菌蛋白质注入宿主细胞中，以促进允许其细胞内生长的吞噬体的生物发生。像许多高度适应的液泡内病原体一样，L。嗜肺菌能够在其吞噬体的内腔中维持中性pH，特别是在感染的早期阶段。然而，在所有情况下，这种观察的分子机制仍然未知。在这份报告中，我们描述了一个军团菌蛋白称为SidK，专门针对主机的v-ATP酶，多亚基机械主要负责在真核细胞中的细胞器酸化的鉴定和表征。我们的研究结果表明，在通过Dot/Icm分泌系统注入感染细胞后，SidK与质子泵的关键组分VatA相互作用。这种结合导致ATP水解和质子转运的抑制。当传递到巨噬细胞时，SidK抑制空泡酸化并损害细胞消化非致病性E.杆菌我们还表明，一个域位于N-末端部分的SidK是负责其与VatA的相互作用。此外，当细菌开始进入新的生长周期时，高度诱导BMPK的表达，这与感染期间其活性的潜在时间要求很好地相关。我们的研究结果表明，分泌蛋白直接靶向v-ATPase构成了L。嗜肺菌，巨噬细胞和阿米巴的空泡病原体。溶酶体的一个标志是低的管腔pH，这对于其成熟以及负责吞噬内容物的有效消化的许多水解酶的活性是重要的。为了在吞噬细胞中生存和复制，成功的细胞内病原体已经进化出各种机制来规避溶酶体杀伤带来的挑战。与细胞内细菌病原体嗜肺军团菌感染相关的一个显著特征是维持含军团菌空泡（LCV）的中性pH，这在感染的早期阶段支持其细胞内生长，而非致病性突变体被认为立即被运输到酸性区室。在真核细胞中，细胞器酸化是由液泡H+-ATP酶介导的，该H +-ATP酶在由ATP水解提供能量的过程中将质子易位到靶区室中。最近发现v-ATP酶与LCV的关联指出了细菌主动调节v-ATP酶活性的必要性。通过筛选L. pneumophila的蛋白质，导致酵母表型类似于它的v-ATPase突变体，我们已经确定了一个底物的L。嗜肺菌Dot/Icm IV型分泌系统，特异性抑制质子转运蛋白的活性。这种蛋白质称为SidK，通过与负责水解ATP的VatA亚基直接相互作用来抑制v-ATP酶的活性。此外，携带SidK的巨噬细胞在吞噬体酸化和溶酶体杀死非病原性细菌方面显示出缺陷。我们还发现，在静止细菌稀释到新鲜培养基中后，SidK的表达被高度诱导，这表明SidK在感染的早期阶段起着重要作用。我们的研究结果揭示了一种机制，通过这种机制，液泡内病原体参与v-ATPase蛋白并抑制其活性，而不是积极避免其与病原体的液泡膜的关联。
Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae. One hallmark of the lysosome is a low luminal pH that is important for its maturation as well as the activity of many hydrolyzing enzymes responsible for efficient digestion of phagocytosed contents. To survive and replicate in phagocytes, successful intracellular pathogens have evolved various mechanisms to circumvent the challenges posed by lysosomal killing. One salient feature associated with infection of the intracellular bacterial pathogen Legionella pneumophila is the maintenance of a neutral pH of the Legionella containing vacuoles (LCVs) that supports its intracellular growth in the early phase of infection, while the nonpathogenic mutants are believed to be immediately trafficked to an acidic compartment. In eukaryotic cells, organelle acidification is mediated by the vacuolar H+-ATPase that translocates protons into target compartments in a process energized by ATP hydrolysis. The recent discovery of the association of v-ATPase with LCVs points to the necessity for active modulation of v-ATPase activity by the bacterium. By screening L. pneumophila proteins that cause a yeast phenotype similar to its v-ATPase mutants, we have identified a substrate of the L. pneumophila Dot/Icm type IV secretion system that specifically inhibits the activity of the proton transporter. This protein, termed SidK, inhibits the activity of v-ATPase by directly interacting with the VatA subunit that is responsible for hydrolyzing ATP. Moreover, macrophages harboring SidK display defects in phagosomal acidification and lysosomal killing of non-pathogenic bacteria. We also found that expression of sidK is highly induced right after stationary bacteria are diluted into fresh medium, suggesting that SidK plays an important role in the early phase of infection. Our results reveal a mechanism by which an intravacuolar pathogen engages the v-ATPase protein and inhibits its activity, rather than actively avoiding its association with the pathogen's vacuolar membrane.
登录
查看更多内容
DOI：
10.1084/jem.158.6.2108
发表时间：
1983-12-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Horwitz MA
通讯作者：
Horwitz MA
影响因子：
3.4
作者：
Amer, AO;Swanson, MS
通讯作者：
Swanson, MS
影响因子：
3.6
作者：
Bardill, JP;Miller, JL;Vogel, JP
通讯作者：
Vogel, JP
影响因子：
7.2
作者：
Kim, W;Wan, CY;Wilkins, TA
通讯作者：
Wilkins, TA
影响因子：
2.6
作者：
GIETZ, RD;SCHIESTL, RH;WOODS, RA
通讯作者：
WOODS, RA