Campbell-Valois François-Xavier, Sachse Martin, Sansonetti Philippe J, Parsot Claude
Institut Pasteur, Ultrapole, Paris, France.
mBio. 2015 May 26;6(3):e02567-14. doi: 10.1128/mBio.02567-14.
The enteropathogenic bacterium Shigella flexneri uses a type 3 secretion apparatus (T3SA) to transfer proteins dubbed translocators and effectors inside host cells, inducing bacterial uptake and subsequent lysis of the entry vacuole. Once in the cytoplasm, the outer membrane protein IcsA induces actin polymerization, enabling cytoplasmic movement and cell-to-cell spread of bacteria. During this infectious process, S. flexneri is targeted by ATG8/LC3. The effector IcsB was proposed to inhibit LC3 recruitment by masking a region of IcsA recognized by the autophagy pathway component ATG5. The effector VirA, a GTPase-activating protein (GAP) for Rab1, was also shown to prevent LC3 recruitment. However, the context of LC3 recruitment around S. flexneri is not fully understood. Here, we show that LC3 is recruited specifically around secreting bacteria that are still present in vacuoles formed during entry and cell-to-cell spread. While LC3 recruitment occurs around a small proportion of intracellular wild-type bacteria, the icsB, virA, and icsB virA mutants display incremental defaults in escape from LC3-positive vacuoles formed during cell-to-cell spread. Our results indicate that IcsB and VirA act synergistically to allow bacteria to escape from LC3-positive vacuoles by acting at or in the immediate vicinity of the vacuole membrane(s). We also demonstrate that LC3 is recruited around bacteria still present in the single-membrane entry vacuole, in a manner akin to that seen with LC3-associated phagocytosis. Our results indicate that LC3 recruitment occurs around bacteria still, or already, in membrane compartments formed during entry and cell-to-cell spread, and not around bacteria free in the cytoplasm.
The targeting of S. flexneri by LC3 is a classic example of the targeting of foreign cytoplasmic particles by autophagy (so-called "xenoautophagy"). It is often assumed that LC3 is recruited around bacteria present in the cytoplasm through the formation of canonical double-membrane autophagosomes. Our results indicate that LC3 is recruited around the entry vacuole composed of a single membrane as in the case of LC3-associated phagocytosis. Effectors IcsB and VirA had been implicated in the blocking of LC3 recruitment, but it was not known if they acted on the same or distinct LC3-recruiting pathways. Our results indicate that LC3 is recruited exclusively around bacteria present in vacuoles formed during entry and cell-to-cell spread and that both IcsB and VirA intervene at the latter stage to facilitate bacterial escape. Our report reconciles several findings and may have broad implications for our understanding of the specific targeting of bacterial pathogens by LC3.
肠道致病菌福氏志贺氏菌利用III型分泌系统（T3SA）将被称为转运蛋白和效应蛋白的蛋白质转移到宿主细胞内，诱导细菌摄取并随后裂解进入液泡。一旦进入细胞质，外膜蛋白IcsA会诱导肌动蛋白聚合，使细菌能够在细胞质中移动并在细胞间传播。在这个感染过程中，福氏志贺氏菌成为自噬相关蛋白ATG8/LC3的作用靶点。有研究提出效应蛋白IcsB通过掩盖自噬途径成分ATG5识别的IcsA区域来抑制LC3的募集。效应蛋白VirA是Rab1的GTP酶激活蛋白（GAP），也被证明可以阻止LC3的募集。然而，福氏志贺氏菌周围LC3募集的具体情况尚未完全了解。在这里，我们表明LC3特异性地募集到仍存在于进入和细胞间传播过程中形成的液泡中的分泌细菌周围。虽然LC3在一小部分细胞内野生型细菌周围募集，但icsB、virA和icsB virA突变体在从细胞间传播过程中形成的LC3阳性液泡中逃逸方面表现出逐渐增加的缺陷。我们的结果表明，IcsB和VirA协同作用，使细菌能够通过作用于液泡膜或其紧邻区域来从LC3阳性液泡中逃逸。我们还证明，LC3以类似于LC3相关吞噬作用的方式募集到仍存在于单膜进入液泡中的细菌周围。我们的结果表明，LC3募集发生在进入和细胞间传播过程中形成的膜隔室内仍存在或已经存在的细菌周围，而不是在细胞质中游离的细菌周围。
LC3对福氏志贺氏菌的靶向作用是自噬（所谓的“异源自噬”）对外源细胞质颗粒靶向作用的经典例子。人们通常认为LC3是通过形成典型的双膜自噬体在细胞质中存在的细菌周围募集的。我们的结果表明，LC3是在由单膜组成的进入液泡周围募集的，就像在LC3相关吞噬作用中一样。效应蛋白IcsB和VirA被认为与阻止LC3募集有关，但尚不清楚它们是否作用于相同或不同的LC3募集途径。我们的结果表明，LC3仅在进入和细胞间传播过程中形成的液泡中存在的细菌周围募集，并且IcsB和VirA都在后期干预以促进细菌逃逸。我们的报告整合了多项研究结果，可能对我们理解LC3对细菌病原体的特异性靶向作用具有广泛的意义。
