Data_Sheet_2_The Temporal Expression of Global Regulator Protein CsrA Is Dually Regulated by ClpP During the Biphasic Life Cycle of Legionella pneumophila.PDF

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, is the causative pathogen of Legionnaires' disease. L. pneumophila adopts a distinct biphasic life cycle that allows it to adapt to environmental conditions for survival, replication, and transmission. This cycle consists of a non-virulent replicative phase (RP) and a virulent transmissive phase (TP). Timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial. Herein, we report evidence that CsrA, a key regulator of the switch between the RP and the TP, is dually regulated in a ClpP-dependent manner during the biphasic life cycle of L. pneumophila. First, we show that the protein level of CsrA is temporal during the life cycle and is degraded by ClpP during the TP. The ectopic expression of CsrA in a ΔclpP mutant, but not in the wild type, inhibits both the initiation of the RP in vitro and the invasiveness to Acanthamoeba castellanii, indicating that the ClpP-mediated proteolytic pathway regulates the CsrA protein level. We further show that the temporally expressed IHFB is the transcriptional inhibitor of csrA and is degraded via a ClpP-dependent manner during the RP. During the RP, the level of CsrA is increased by promoting the degradation of IHFB and reducing the degradation of the accumulated CsrA via a ClpP-dependent manner. During the TP, the level of CsrA is decreased by inhibiting the degradation of IHFB and promoting the degradation of the accumulated CsrA via a ClpP-dependent manner as well. In conclusion, our results show that the growth-stage-specific expression level of CsrA is dually regulated by ClpP-dependent proteolysis at both the transcription and protein levels during the biphasic life cycle of L. pneumophila.

嗜肺军团菌（Legionella pneumophila）是一种寄生于原生动物（protozoa）的环境细菌，亦是军团病（Legionnaires' disease）的致病病原体。该菌拥有独特的双相生命周期（biphasic life cycle），可使其适应不同环境条件以完成存活、增殖与传播过程，该周期分为非毒力复制期（RP，non-virulent replicative phase）与毒力传播期（TP，virulent transmissive phase）两个阶段。以生命周期依赖的方式对生长与毒力因子进行适时且精细的表达调控，是其完成生命周期的关键环节。本文报道了嗜肺军团菌双相生命周期中，作为复制期与传播期转换关键调控因子的CsrA（CsrA）以ClpP（ClpP）依赖的方式受到双重调控的实验证据。首先，我们发现CsrA的蛋白水平在生命周期中呈时序性变化，并在毒力传播期被ClpP降解。在ΔclpP突变株（ΔclpP mutant）中异位表达（ectopic expression）CsrA，可同时抑制体外非毒力复制期的启动以及对卡氏棘阿米巴（Acanthamoeba castellanii）的侵袭能力，而在野生型（wild type）菌株中则无此效应，这表明ClpP介导的蛋白水解通路（proteolytic pathway）可调控CsrA的蛋白水平。进一步研究证实，时序表达的IHFB（IHFB）是csrA的转录抑制剂（transcriptional inhibitor），并在非毒力复制期以ClpP依赖的方式被降解。在非毒力复制期，CsrA的水平通过两种途径得以提升：一是促进IHFB的降解，二是通过ClpP依赖的方式减少已积累的CsrA的降解；而在毒力传播期，CsrA的水平则通过相反机制降低：一是抑制IHFB的降解，二是同样以ClpP依赖的方式促进已积累的CsrA的降解。综上，本研究结果表明，在嗜肺军团菌的双相生命周期中，CsrA的生长阶段特异性表达水平，在转录与蛋白两个层面均受到ClpP依赖的蛋白水解作用的双重调控。