Data Sheet 1_Two cinnamoyl hydroxamates as potential quorum sensing inhibitors against Pseudomonas aeruginosa.docx

IntroductionPseudomonas aeruginosa is a ubiquitous pathogen that causes various infectious diseases through the regulation of quorum sensing (QS). The strategy of interfering with the QS systems of P. aeruginosa, coupled with a reduction in the dosage of conventional antibiotics, presents a potential solution to treating infection and mitigating antibiotic resistance. In this study, seven cinnamoyl hydroxamates were synthesized to evaluate their inhibitory effects on QS of P. aeruginosa. Among these cinnamic acid derivatives, we found cinnamoyl hydroxamic acid (CHA) and 3-methoxy-cinnamoyl hydroxamic acid (MCHA) were the two most effective candidates. Furtherly, the effect of CHA and MCHA on the production of virulence factors and biofilm of P. aeruginosa were evaluated. Ultimately, our study may offer promising potential for treating P. aeruginosa infections and reducing its virulence.MethodsThe disc diffusion test were conducted to evaluate inhibitory effects on QS of seven cinnamoyl hydroxamates. The influence of CHA and MCHA on the production of virulence and flagellar motility of P. aeruginosa was furtherly explored. Scanning electron microscopy (SEM) experiment were conducted to evaluate the suppression of CHA and MCHA on the formed biofilm of P. aeruginosa. RT-qPCR was used to detect rhlI, lasA, lasB, rhlA, rhlB, and oprL genes in P. aeruginosa. In silico docking study was performed to explore the molecular mechanism of CHA and MCHA. The synergistic effects of CHA with gentamicin were detected on biofilm cell dispersal.ResultAfter treatment of CHA or MCHA, the production of multiple virulence factors, including pyocyanin, proteases, rhamnolipid, and siderophore, and swimming and swarming motilities in P. aeruginosa were inhibited significantly. And our results showed CHA and MCHA could eliminate the formed biofilm of P. aeruginosa. RT-qPCR revealed that CHA and MCHA inhibited the expression of QS related genes in P. aeruginosa. Molecular docking indicated that CHA and MCHA primarily inhibited the RhlI/R system in P. aeruginosa by competing with the cognate signaling molecule C4-HSL.Additionally, CHA exhibited potent synergistic effects with gentamicin on biofilm cell dispersal.DiscussionP. aeruginosa is one of the most clinically and epidemiologically important bacteria and a primary cause of catheter-related urinary tract infections and ventilator-associated pneumonia. This study aims to explore whether cinnamoyl hydroxamates have inhibitory effects on QS. And our results indicate that CHA and MCHA, as two novel QSIs, offer promising potential for treating P. aeruginosa infections and reducing its virulence.

铜绿假单胞菌作为一种广泛存在的病原体，通过调节群体感应（QS）机制引发多种感染性疾病。干扰铜绿假单胞菌的QS系统，并结合降低传统抗生素的用量，为治疗感染和缓解抗生素耐药性提供了一种潜在的解决方案。在本研究中，合成了七种肉桂酰羟基甲酸酯以评估其对铜绿假单胞菌QS抑制效果。在这些肉桂酸衍生物中，我们发现了肉桂酰羟基甲酸（CHA）和3-甲氧基肉桂酰羟基甲酸（MCHA）是两种最为有效的候选化合物。进一步地，我们评估了CHA和MCHA对铜绿假单胞菌致病因子和生物膜形成的影响。最终，本研究可能为治疗铜绿假单胞菌感染和降低其致病力提供了有希望的潜力。

方法：通过纸片扩散法评估了七种肉桂酰羟基甲酸酯对QS的抑制作用。进一步探讨了CHA和MCHA对铜绿假单胞菌致病性和鞭毛游动性的影响。扫描电子显微镜（SEM）实验用于评估CHA和MCHA对形成的铜绿假单胞菌生物膜的抑制作用。实时定量PCR（RT-qPCR）用于检测铜绿假单胞菌中的rhlI、lasA、lasB、rhlA、rhlB和oprL基因。通过计算机模拟对接研究，探索了CHA和MCHA的分子机制。检测了CHA与庆大霉素的协同作用对生物膜细胞分散的影响。

结果：经CHA或MCHA处理后，铜绿假单胞菌中多种致病因子（包括青霉素、蛋白酶、鼠李糖脂和铁载体）以及游动性和群体游动性均受到显著抑制。我们的结果表明，CHA和MCHA能够消除形成的铜绿假单胞菌生物膜。RT-qPCR显示，CHA和MCHA抑制了铜绿假单胞菌中QS相关基因的表达。分子对接表明，CHA和MCHA主要通过竞争同源信号分子C4-HSL，主要抑制铜绿假单胞菌中的RhlI/R系统。此外，CHA与庆大霉素在生物膜细胞分散方面表现出显著的协同作用。

讨论：铜绿假单胞菌是临床上和流行病学上最重要的细菌之一，是导管相关性尿路感染和呼吸机相关性肺炎的主要致病菌。本研究旨在探讨肉桂酰羟基甲酸酯是否具有对QS的抑制作用。我们的结果指示，作为两种新型QS抑制剂（QSIs），CHA和MCHA在治疗铜绿假单胞菌感染和降低其致病力方面具有有希望的潜力。