Role of autocleavage in the function of a type III secretion specificity switch protein in Salmonella enterica serovar Typhimurium.
Role of autocleavage in the function of a type III secretion specificity switch protein in Salmonella enterica serovar Typhimurium.
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DOI：
10.1128/mbio.01459-15
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发表时间：
2015-10-13
期刊：
影响因子：
6.4
通讯作者：
Wagner S
中科院分区：
文献类型：
--
作者：
Monjarás Feria JV;Lefebre MD;Stierhof YD;Galán JE;Wagner S
III 型分泌系统 (T3SS) 是许多革兰氏阴性细菌用来将细菌效应蛋白注入真核宿主细胞以促进细菌存活和定殖的多蛋白机器。 T3SS 的核心单元是针状复合体，这是一种介导分泌蛋白穿过细菌包膜的超分子结构。 T3SS 的一个显着特征是蛋白质输出以严格的分层方式发生，其中首先分泌注定形成针状复合体丝和相关结构的蛋白质，然后分泌效应子和促进其通过目标宿主细胞膜易位的蛋白质。分泌层次结构是通过复杂的机制建立的，涉及多个 T3SS 相关成分，包括“开关蛋白”，一种高度保守的内膜蛋白酶，可进行自催化裂解。有人提出，开关蛋白的自动切割是底物转换的触发因素。我们在此表明，肠沙门氏菌血清型鼠伤寒开关蛋白 SpaS 的自动裂解是一个不受调控的过程，发生在其折叠之后和掺入针复合物之前。与预先切割的 SpaS 形式组装的针复合物的功能方式与野生型形式没有区别。此外，由外部蛋白酶加工的 SpaS 工程突变体也显示出野生型功能。这些结果表明，裂解事件本身并不提供底物转换信号，但支持以下假设：裂解使 SpaS 形成正确的构象，使其能够发挥其转换功能。细菌与真核宿主的相互作用通常涉及用于靶向递送细菌效应蛋白的复杂分子机器。一些革兰氏阴性细菌的 III 型分泌系统就是这样一种机器，用于将多种结构多样的细菌蛋白注入宿主细胞。这些系统功能的关键是它们以严格的层次顺序分泌蛋白质的能力，但目前尚不清楚转换机制是如何工作的。转换机制的核心是高度保守的内膜蛋白酶，它会进行自催化裂解。尽管之前有人建议自动裂解事件是底物切换的触发因素，但我们在这里表明情况并非如此。相反，我们的结果表明，切割使蛋白质具有正确的构象，使其能够发挥其转换功能。这些发现可能有助于开发 III 型分泌机抑制剂，为治疗各种传染病提供新的治疗途径。
Type III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of the Salmonella enterica serovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage event per se does not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function. Bacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.