A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells.
A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells.
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DOI：
10.1371/journal.ppat.1006354
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发表时间：
2017-04
期刊：
影响因子：
6.7
通讯作者：
Steele-Mortimer O
中科院分区：
文献类型：
--
作者：
Finn CE;Chong A;Cooper KG;Starr T;Steele-Mortimer O
肠病原体鼠伤寒沙门氏菌使用III型分泌系统1(T3SS1)在肠道内建立感染。该系统跨质膜转运的效应蛋白促进了肠上皮细胞的侵袭。肌醇磷酸酶SopB就是一种这样的效应因子，它有助于侵袭并介导促生存激酶Akt的激活。在内化后，一些细菌从含有沙门氏菌的液泡逃逸到细胞质中，有证据表明T3SS1在这个亚群中表达。在这里，我们使用SopB作为模型效应器，研究了T3SS1在入侵后的作用。在培养的上皮细胞中，SopB依赖的Akt磷酸化在感染的两个不同阶段被观察到：侵袭期间和侵袭后即刻，以及后来的胞浆复制高峰期。单细胞分析表明，胞质沙门氏菌通过T3SS1传递SopB。虽然SopB缺失突变体的细胞内复制没有受到影响，但感染ΔSopB的细胞表现出缺乏Akt磷酸化，死亡时间提前，裂解增加。当SopB在胞浆沙门氏菌中被特异性诱导表达时，这些效应恢复到在WT感染细胞中观察到的水平，表明第二波SopB通过Akt激活保护该感染群体免受细胞死亡。因此，在上皮细胞定植过程中，T3SS1在时间上有两个不同的作用。此外，我们发现，胞质细菌对SopB的转运是不依赖于转位的，而典型的效应器跨真核细胞膜的转位需要形成转位子孔。对于另一种T3SS1效应器SIPA，也观察到了这种机制。这些发现揭示了T3SS的功能和机械适应性，可以在不同的微环境中利用。非伤寒沙门氏菌是世界范围内食源性疾病的重要病原体。这些兼性细胞内细菌使用一种特殊的III型分泌系统(T3SS1)来入侵肠道上皮细胞。通过该系统跨真核细胞质膜转运的效应蛋白可诱导肌动蛋白重排和靶向信号通路。SopB就是一种这样的效应因子，它有助于侵袭并介导促生存激酶Akt的激活。在上皮细胞内，沙门氏菌在修改后的吞噬小体内生存和复制，称为含有沙门氏菌的液泡或宿主细胞胞浆。在这里，我们使用SopB作为模型效应器，研究了T3SS1在上皮细胞侵袭后的作用。SopB依赖的Akt磷酸化在感染的两个不同阶段被观察到：侵袭期间和侵袭后立即，以及后来的胞浆复制高峰期。胞质沙门氏菌转导SopB需要T3SS1，但不依赖于转位。对另一种T3SS1效应器SIPA也观察到了这一点，表明T3SS1效应器可能直接分泌到细胞质中。SopB缺失突变体的感染消除了Akt磷酸化的诱导，缩短了感染细胞的寿命。通过在胞浆细菌中特异性地表达SopB，这些效应被逆转，证实了SopB和T3SS1在感染的胞浆阶段的作用。因此，T3SS1在上皮细胞定植过程中有两个不同的作用。
Type III secretion system 1 (T3SS1) is used by the enteropathogen Salmonella enterica serovar Typhimurium to establish infection in the gut. Effector proteins translocated by this system across the plasma membrane facilitate invasion of intestinal epithelial cells. One such effector, the inositol phosphatase SopB, contributes to invasion and mediates activation of the pro-survival kinase Akt. Following internalization, some bacteria escape from the Salmonella-containing vacuole into the cytosol and there is evidence suggesting that T3SS1 is expressed in this subpopulation. Here, we investigated the post-invasion role of T3SS1, using SopB as a model effector. In cultured epithelial cells, SopB-dependent Akt phosphorylation was observed at two distinct stages of infection: during and immediately after invasion, and later during peak cytosolic replication. Single cell analysis revealed that cytosolic Salmonella deliver SopB via T3SS1. Although intracellular replication was unaffected in a SopB deletion mutant, cells infected with ΔsopB demonstrated a lack of Akt phosphorylation, earlier time to death, and increased lysis. When SopB expression was induced specifically in cytosolic Salmonella, these effects were restored to levels observed in WT infected cells, indicating that the second wave of SopB protects this infected population against cell death via Akt activation. Thus, T3SS1 has two, temporally distinct roles during epithelial cell colonization. Additionally, we found that delivery of SopB by cytosolic bacteria was translocon-independent, in contrast to canonical effector translocation across eukaryotic membranes, which requires formation of a translocon pore. This mechanism was also observed for another T3SS1 effector, SipA. These findings reveal the functional and mechanistic adaptability of a T3SS that can be harnessed in different microenvironments. Non-Typhoidal Salmonella are important agents of food borne disease worldwide. These facultative intracellular bacteria use a specialized Type III Secretion (T3SS1) system to invade intestinal epithelial cells. Effector proteins translocated by this system across the eukaryotic plasma membrane induce actin rearrangements and target signaling pathways. One such effector is SopB, which contributes to invasion and mediates activation of the pro-survival kinase Akt. Within epithelial cells, Salmonella survive and replicate within a modified phagosome, known as the Salmonella-containing vacuole, or the host cell cytosol. Here, we investigated the post-invasion role of T3SS1 in epithelial cells, using SopB as a model effector. SopB-dependent Akt phosphorylation was observed at two distinct stages of infection: during and immediately after invasion, and later during peak cytosolic replication. SopB delivery by cytosolic Salmonella required T3SS1 but was translocon-independent. This was also observed for another T3SS1 effector, SipA, indicating that T3SS1 effectors may be secreted directly into the cytosol. Infection with a SopB deletion mutant eliminated the induction of Akt phosphorylation and decreased the lifespan of infected cells. These effects were reversed by expressing SopB specifically in cytosolic bacteria, confirming a role for SopB and T3SS1 during the cytosolic stage of infection. Thus, T3SS1 has two temporally distinct roles during epithelial cell colonization.