Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospitalgrid.32224.35, Boston, Massachusetts, USA.
Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA.
mBio. 2021 Dec 21;12(6):e0302121. doi: 10.1128/mBio.03021-21. Epub 2021 Nov 23.
spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled cross-linking by membrane-permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on posttranslational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by during infection. Type 3 secretion systems are nanomachines employed by many bacteria, including , which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by Shigella flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during infection.
spp. 是引起细菌性痢疾的人类细菌病原体。毒力取决于 III 型分泌系统 (T3SS)，这是一种存在于多种重要人类和植物病原体中的高度保守结构。一旦与宿主细胞接触，T3SS 转位器就会被递送到宿主膜上，促进细菌与膜的对接，并使效应蛋白进入宿主细胞质。转位器由两个蛋白质组成，IpaB 和 IpaC，它们共同形成宿主质膜内的这种多聚体结构。当 IpaC 与宿主中间丝相互作用时，转位器会发生构象变化，允许细菌与转位器对接，并与宿主肌动蛋白聚合一起，使随后的效应蛋白通过转位器孔进行易位。为了更深入地了解转位器，我们使用半胱氨酸取代突变与定点标记和膜透性巯基反应试剂的邻近交联相结合，在嵌入质膜的孔中绘制了 IpaB 的拓扑结构。我们证明 IpaB 的功能依赖于质粒编码的酰基载体蛋白的翻译后修饰。我们表明 IpaB 的第一个跨膜结构域排列在转位器孔道的内部，使得孔道的 IpaB 部分形成一个漏斗状形状，通向宿主细胞质。此外，我们还确定了 IpaB 胞质结构域内的一些区域，这些区域突出到并与孔道紧密相关。总之，这些结果为 Shigella flexneri 天然递送至感染期间的转位器中 IpaB 的排列方式提供了一个框架。III 型分泌系统是许多细菌使用的纳米机器，包括 ，它将细菌毒力蛋白递送到人类细胞中，以改变促进感染的细胞功能。 通过 IpaB 和 IpaC 蛋白在人类细胞膜中形成的孔来输送 毒力蛋白。在这里，我们定义了 IpaB 如何有助于 Shigella flexneri 天然递送至人细胞膜中的孔的形成。我们表明，IpaB 的一个特定结构域（跨膜结构域 1）排列在大部分孔道中，而位于人类细胞内部的 IpaB 部分则回折到孔道内或与孔道紧密相关。这些发现为了解在 Shigella flexneri 感染过程中作为输送毒力蛋白进入人类细胞的管道的孔的组织和功能提供了新的见解。
