Department of Molecular Biology, Princeton Universitygrid.16750.35, Princeton, New Jersey, USA.
Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
mBio. 2022 Oct 26;13(5):e0155922. doi: 10.1128/mbio.01559-22. Epub 2022 Sep 8.
Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor RhlR, an interaction that enhances the affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, the virulence function of PqsE can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing enhanced green fluorescent protein-PqsE fusions to define the protein interactomes of wild-type PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wild-type PqsE while not forming associations with PqsE(D73A). In the Δ strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase for RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing. Pseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that, to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins, namely, PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates, which will be investigated for potential roles in pathogenesis.
铜绿假单胞菌是一种人类病原体，依赖群体感应来建立感染。PqsE 群体感应蛋白是铜绿假单胞菌毒力因子产生和感染所必需的。PqsE 在群体感应自动诱导剂 PQS 的生物合成中具有报道的酶功能。然而，这种活性是多余的，因为在没有 PqsE 的情况下，这个角色由替代硫酯酶来完成。相反，PqsE 通过与群体感应受体/转录因子 RhlR 的蛋白质-蛋白质相互作用来驱动铜绿假单胞菌的致病特征，这种相互作用增强了 RhlR 对靶 DNA 序列的亲和力。PqsE 的催化活性对于与 RhlR 的相互作用不是必需的。因此，PqsE 的毒力功能可以与其催化功能分离。在这里，我们提出了一种免疫沉淀-质谱方法，该方法使用增强型绿色荧光蛋白-PqsE 融合蛋白来定义野生型 PqsE 和无催化活性的 PqsE(D73A)变体在铜绿假单胞菌中的蛋白质相互作用组及其对 RhlR 的依赖性。鉴定出几种与野生型 PqsE 具有特异性相互作用而不与 PqsE(D73A)形成关联的蛋白质。在Δ菌株中，鉴定出更多特定的 PqsE 相互作用物，包括 RhlR 的伴侣自动诱导物合酶，称为 RhlI。总的来说，这些结果表明，特定的蛋白质-蛋白质相互作用取决于 PqsE 的催化活性，并且 RhlR 可能阻止蛋白质与 PqsE 相互作用，可能是由于 RhlR 和其他蛋白质之间对 PqsE 结合的竞争。我们的结果为鉴定 PqsE 的催化功能以及可能涉及铜绿假单胞菌群体感应的新蛋白质提供了基础。铜绿假单胞菌会导致易感染人群（包括免疫功能低下的个体、烧伤患者和接受化疗的癌症患者）发生医院感染。对于铜绿假单胞菌感染，目前尚无有效的治疗方法，而且这种感染通常对广泛的抗生素有抗药性。动物模型表明，为了建立感染并导致疾病，铜绿假单胞菌依赖于两种蛋白质（即 PqsE 和 RhlR）之间的相互作用。可能还有涉及 PqsE 的其他蛋白质-蛋白质相互作用，如果确定，可能会被用于设计新的治疗策略来对抗铜绿假单胞菌。在这里，我们揭示了 PqsE 参与的以前未知的蛋白质相互作用，将对其在发病机制中的潜在作用进行研究。
