Almagro-Moreno Salvador, Kim Tae K, Skorupski Karen, Taylor Ronald K
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.
PLoS Genet. 2015 Apr 7;11(4):e1005145. doi: 10.1371/journal.pgen.1005145. eCollection 2015 Apr.
Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state.
霍乱弧菌O1是水生环境中的天然居民，可引发腹泻疾病霍乱。其两个主要的毒力调节因子TcpP和ToxR定位于内膜。TcpP编码于霍乱弧菌致病岛（VPI）上，这是一个水平获得的可移动遗传元件，主要在毒力基因调控中发挥作用。已表明TcpP会响应不利于毒力基因表达的环境条件而经历受调控的膜内蛋白水解（RIP）。ToxR编码于祖先基因组中，存在于霍乱弧菌的非致病菌株中，表明它在人类宿主之外也有作用。在本研究中，我们表明在碱性pH值下的营养限制条件下（这是生长稳定期出现的一种情况），霍乱弧菌中的ToxR会经历RIP。这个过程涉及位点2蛋白酶RseP（YaeL），并且依赖于RpoE介导的周质应激反应，因为编码这两种蛋白质的基因的缺失突变体在碱性pH值下的营养限制条件下不能对ToxR进行蛋白水解。我们确定，通过基因手段或蛋白水解导致的ToxR缺失与霍乱弧菌进入一种休眠状态有关，在这种状态下，细菌通常存在于水生环境中，称为活的但不可培养（VBNC）状态。能够对ToxR进行蛋白水解或不编码ToxR的菌株失去可培养性，经历与VBNC细胞相关的形态变化，但在碱性pH值下的营养限制条件下仍保持存活。另一方面，不能对ToxR进行蛋白水解的突变菌株仍可培养，并保持处于活跃生长状态的细胞形态。总体而言，我们的研究结果提供了一个毒力调节因子的蛋白水解与病原体进入环境持久状态之间的联系。
