The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.

针对环境变化，铜绿假单胞菌（Pseudomonas aeruginosa）可从浮游（planktonic，自由游动）生活方式转换为固着（sessile，生物被膜）生活方式。双组分系统（two-component system, TCS）GacS/GacA可激活两种小型非编码RNA（small non-coding RNAs）RsmY和RsmZ的生成，但有四种组氨酸激酶（histidine kinases, HKs）——RetS、GacS、LadS及PA1611——在该过程中发挥关键作用。杂合组氨酸激酶（hybrid HK）RetS可通过形成异二聚体（heterodimer），阻断非典型组氨酸激酶GacS的自身磷酸化（autophosphorylation）。结构上与GacS同源的杂合组氨酸激酶PA1611，在铜绿假单胞菌中与RetS的互作方式与GacS极为相似。杂合组氨酸激酶LadS可通过一种尚未阐明的机制，在表型上拮抗RetS的功能。这四种感应蛋白在大多数假单胞菌属物种中均存在，但其特性及信号传导模式可能因物种而异。本研究在铜绿假单胞菌中证实，LadS可调控rsmY与rsmZ的基因表达，且该调控过程依赖GacS/GacA TCS。我们还发现，与RetS不同，LadS无需与GacS或RetS形成异二聚体，即可通过GacS/GacA TCS传导信号。进一步研究表明，LadS依赖真正的磷酸接力（phosphorelay）通路发挥作用，该通路同时需要LadS的传递结构域与接收结构域参与。LadS的信号传导最终需要GacS的备用组氨酸磷酸转移结构域（alternative histidine-phosphotransfer domain, Hpt）作为杂合激酶的磷酸接力中继位点。因此，LadS组氨酸激酶与GacS/GacA TCS共同构成了一套具有独特磷酸接力级联反应的多组分信号转导系统，即H1_LadS→D1_LadS→H2_GacS→D2_GacA。这揭示了一种独特的调控策略：通过复杂的多感应网络调控单一输出目标——即小RNA（sRNA）水平的调控——以此精准适配适应性生物被膜形成与毒力响应。