霍乱弧菌是引起腹泻病霍乱的革兰氏阴性胃肠道病原体。关键毒力因子的表达,霍乱毒素和毒素共同调节的菌毛,直接由ToxT调节,间接由两个跨膜转录调节因子(TTRs)调节,ToxR和TcpP,促进toxT的表达。TcpP丰度和活性受TcpH控制,一种单程跨膜蛋白,它保护TcpP免受两步蛋白水解过程的影响,称为调节膜内蛋白水解(RIP)。TcpH介导的TcpP保护机制代表了我们对霍乱弧菌发病机理的理解的主要空白。tcpH的缺乏导致TcpP在体外的无阻碍降解和在霍乱弧菌定植的新生小鼠模型中的定植缺陷。这里,我们表明TcpH通过直接相互作用保护TcpP免受RIP的影响。我们还证明了α-亚麻酸,一种膳食脂肪酸,在需要TcpH跨膜结构域的机制中,通过TcpP和TcpH分子在洗涤剂抗性膜(DRMs)内的共缔合来促进对RIP的TcpH依赖性抑制。一起来看,我们的数据支持一个模型,其中霍乱弧菌细胞使用外源α-亚麻酸在体内重塑磷脂双层,导致TcpP和TcpH在DRMs内共结合,其中TcpP的RIP被TcpH抑制,从而促进霍乱弧菌的致病性。
目的:霍乱弧菌继续对全球健康构成重大负担,需要采用替代治疗方法,由于不断发展的多药耐药菌株。toxT的转录,在TcpP和ToxR的刺激下,对霍乱弧菌的发病机制至关重要。我们的结果表明,TcpP,toxT基因表达的主要调节因子之一,被TcpH保护免受蛋白水解,通过直接互动。此外,我们发现了一种肠道代谢产物,α-亚麻酸,刺激TcpP和TcpH在耐去污剂膜(也称为脂质有序膜结构域)内的联合,从而支持TcpP蛋白水解的TcpH依赖性拮抗作用。这里提供的数据扩展了我们对RIP的了解,霍乱弧菌的毒力基因调控,and,据我们所知,提供了第一个证据证明霍乱弧菌中存在脂质有序膜。这里提出的模型还表明,TTR,在细菌和古细菌中很常见,和肠杆菌中存在的共成分信号转导系统,也可能受到类似的影响。
Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key
virulence factors, cholera toxin and toxin-coregulated pilus, is regulated directly by ToxT and indirectly by two transmembrane transcription regulators (TTRs), ToxR and TcpP, that promote the expression of toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH-mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. The absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that α-linolenic acid, a dietary fatty acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs) in a mechanism requiring the TcpH transmembrane domain. Taken together, our data support a model where V. cholerae cells use exogenous α-linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity.
OBJECTIVE: Vibrio cholerae continues to pose a significant global burden on health and an alternative therapeutic approach is needed, due to evolving multidrug resistance strains. Transcription of toxT, stimulated by TcpP and ToxR, is essential for V. cholerae pathogenesis. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction. Furthermore, we identified a gut metabolite, α-linolenic acid, that stimulates the co-association of TcpP and TcpH within detergent-resistant membranes (also known as lipid-ordered membrane domains), thereby supporting TcpH-dependent antagonism of TcpP proteolysis. Data presented here extend our knowledge of RIP,
virulence gene regulation in V. cholerae, and, to the best of our knowledge, provides the first evidence that lipid-ordered membranes exist within V. cholerae. The model presented here also suggests that TTRs, common among bacteria and archaea, and co-component signal transduction systems present in Enterobacteria, could also be influenced similarly.