Unravelling the Genome-Wide Contributions of Specific 2-Alkyl-4-Quinolones and PqsE to Quorum Sensing in <i>Pseudomonas aeruginosa</i>

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl-4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response.pqs群体感应（Quorum Sensing, QS）系统在体外及感染动物模型中对铜绿假单胞菌（Pseudomonas aeruginosa）的毒力至关重要，被视为抗毒力制剂开发的理想靶点。然而，这一复杂QS回路的各个独立组分在毒力调控中的确切作用仍有待阐明。pqs QS系统的关键组分包括2-庚基-4-羟基喹啉（2-heptyl-4-hydroxyquinoline, HHQ）、2-庚基-3-羟基-4-喹诺酮（2-heptyl-3-hydroxy-4-quinolone, PQS）、2-庚基-4-羟基喹啉N-氧化物（2-heptyl-4-hydroxyquinoline N-oxide, HQNO）、转录调控因子PqsR以及PQS效应元件PqsE。为明确上述各组分在QS介导的基因调控中的独立贡献，研究人员对经过工程改造的铜绿假单胞菌菌株开展了转录组分析并进行验证：这些菌株的2-烷基-4-喹诺酮（2-alkyl-4-quinolones, AQs）生物合成途径与pqsE、pqsR的表达已被解偶联，该设计便于识别单个pqs系统组分所调控的基因。所得结果表明：① PQS生物合成前体HHQ可触发依赖于PqsR的正反馈环路，仅能促进pqsABCDE操纵子的表达上调；② PqsE参与调控编码核心毒力决定因子及生物被膜形成相关的众多基因；③ PQS可通过依赖于PqsR以及不依赖于PqsR的两条途径，促进AQs的生物合成、铁饥饿响应相关基因的表达以及毒力因子的产生；④ HQNO不会对转录产生影响，因此无法作为QS信号分子发挥功能。总体而言，本研究明确了单个pqs系统组分所调控的特定调控子（regulon），并揭示了PQS可独立于其激活PqsR以及诱导铁饥饿响应的能力之外，参与基因调控的功能。