病原生物膜利用工程定额淬Lactonases防有毒中断
Summary
Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.
Abstract
The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm’s tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.
Introduction
对于感染性疾病的治疗方案已被复杂化在多药耐药性细菌是免疫广泛的抗生素药物1的快速增加。从耐药细菌介导的感染的高发病率和死亡率，有必要升级药物开发过程和/或探索更好抗菌替代改善的治疗选择。近来，抗毒性的方法是获得给出其在经由非杀菌方法防止致病潜力的兴趣，因此减轻耐药机制2的风险。
群体感应是细菌毒力和这种信号中断现象的一个“总开关”是对发病3一个有前途的抗毒性的方法。毒力的发作需要仲裁分子在细胞外环境中的积累达到临界细菌种群密度之后。由于现状朗姆酒分子扩散回进入胞内基质中，与它们的同源受体结合导致的毒力因子的活化，以及与抗生素抗性和生物膜形成4相关的基因。在一般情况下，群体感应中断涉及抑制仲裁分子和受体相互作用，而不会影响主代谢途径。因此，它不具有对细胞生长的任何直接含意。由于健康不受损害，是最小的选择压力细菌进化并获得对这种治疗5阻力。此外，群体感应中断可以与固有的细菌保护机制干扰，如在生物膜形成，它提供了保护从抗菌剂和宿主免疫反应的情况下。
据估计，在复杂的生物膜状矩阵存在99％地球上的微生物，赋予关键存活优势居住在第微生物ESE结构6。更重要的是，形成这些无柄域是最持久和慢性医院获得性感染7的原因。 鲍曼不动杆菌是一个与全球医院获得性感染相关联，其毒力在很大程度上归因于群体感应的主要的人类病原体之一介导的生物膜形成8。仲裁淬酶已被成功地用于通过靶向一组化合物已知为 N -酰基高丝氨酸内酯（信号分子）是由革兰氏阴性细菌产生的9扰乱仲裁介导的信号转导。一些研究也扩大于使用这些酶通过毒力因子的表达和细胞数在生物膜10,11减少到方框细菌发病机理。不幸的是，仍缺乏有效利用仲裁 – 淬火酶对生物膜形成由细菌病原体扪示范的。该重新已经尝试使用定额抑制剂（AHL类似物）代替法定淬火酶，以破坏A.鲍曼不动杆菌生物膜形成12。虽然使用小分子抑制剂的这种方法是一种有效的方法，维持在平利用其生物利用度可能是一个挑战。与此相反，使用催化仲裁 – 淬火酶可以规避生物利用度问题，因为酶是朝固定上用于治疗效果的生物医学装置的表面更适合。
在这里，我们描述了从地芽孢kaustophilus（GKL）13对细菌生物膜的形成工程定额淬火lactonases的影响进行评估，采用结晶紫染色和激光共聚焦扫描显微镜（CLSM）。这项研究是生物膜分裂的在一个临床相关的A中的第一次成功的示范鲍曼不动杆菌菌株S1采用定额淬火酶。的方法中所述在这项研究中的用于评估对致病性革兰氏阴性细菌后续治疗的努力发展其他仲裁淬火酶的效力是有用的。
Protocol
Representative Results
Discussion
在这两组实验中，A杆菌 S1中培养在LB培养基无氯化钠作为高盐浓度可减少生物膜由细菌15形成的量。这种伪影的存在可能会低估生物膜的形成的量，以及仲裁 – 淬火酶在不同的处理条件的影响。使用催化失活酶是重要的，作为阴性对照，以消除酶封存的可能影响。 图1表明，即使不活动酶螯合仲裁分子，生物膜的形成不被中断。
鲍曼不动杆菌</em…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Academic Research Fund of the Ministry of Education, and the National Medical Research Council and the National Research Foundation, Singapore.
Materials
| Tryptone | BD | 211705 | |
| Yeast Extract | BD | 212750 | |
| 96-well plate | Costar | 3596 | |
| Crystal Violet | Sigma-Aldrich | C6158 | |
| Acetic Acid | Lab-Scan | PLA00654X | Caution: Flammable |
| μ-Dish | Ibidi | 80136 | |
| Alex Fluo 488-conjugated WGA | Invitrogen | W11261 | |
| Hank’s balanced salt solution | Invitrogen | 141475095 | |
| Formaldehyde | Sigma-Aldrich | F8775 | Caution: Corrosive |
| Synergy HT Microplate Reader | BioTek | ||
| 1X-81 Inverted Fluorescence Microscope | Olympus |
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