Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

摘要

群体淬灭酶是抗毒力和抗菌选择，通过阻止与抗生素耐药性和生物膜形成相关的毒力因子和基因的表达，可以减轻发病机制而不会产生耐药性的风险。在这项研究中，我们报道了一种方法，该方法证明了群体猝灭酶在细菌生物膜破坏中的功效。

摘要

多重耐药细菌的迅速出现加速了对新型治疗方法的需求，以对抗危及生命的感染。细菌感染的持续性通常与群体感应介导的生物膜形成有关。因此，该信号回路的破坏提供了一种有吸引力的抗毒力策略。据报道，群体猝灭内酯酶是群体感应回路的有效破坏物。然而，关于这些酶有效利用细菌生物膜形成的报道很少。该方案描述了一种通过使用工程化的群体淬灭内酯酶来破坏临床相关的鲍曼不动杆菌 S1 菌株中生物膜形成的方法。鲍曼不动杆菌是一种与全球严重医院获得性感染有关的主要人类病原体，其毒力主要归因于其生物膜的顽强性。工程化内酯酶处理实现了鲍曼不动杆菌 S1 生物膜的显著减少。这项研究还显示了未来使用工程化群体猝灭酶治疗生物膜介导的细菌疾病的可能性。最后，该方法可用于评估有前途的群体猝灭酶的能力。

引言

Treatment options for infectious diseases have been complicated by the rapid increase in multidrug-resistant bacteria that are immune to a wide range of antibiotic drugs¹. With high morbidity and mortality rates from resistant bacteria-mediated infections, there is a need to escalate drug development processes and/or explore better anti-bacterial alternatives to improve therapeutic options. Lately, the anti-virulence approach is gaining interest given its potential in preventing virulence via non-bactericidal methods, hence mitigating the risks of resistance mechanisms².

Quorum-sensing is a 'master switch' in bacterial virulence and disruption of this signaling phenomenon is a promising anti-virulence method against pathogenesis³. The onset of virulence requires the accumulation of quorum molecules in the extracellular environment after a critical bacterial population density is reached. As quorum molecules diffuse back into the intracellular matrix, binding with their cognate receptors leads to the activation of virulence factors as well as genes associated with antibiotic resistance and biofilm formation⁴. In general, quorum-sensing disruption involves inhibiting quorum molecule and receptor interaction without affecting primary metabolic pathways. Hence, it does not have any direct implication on cellular growth. Since fitness is not compromised, there is minimal selection pressure for bacteria to evolve and gain resistance against such treatments⁵. In addition, quorum-sensing disruption can interfere with inherent bacterial protective mechanisms, as in the case of biofilm formation, which provides protection from anti-bacterial agents and host immune responses.

It is estimated that 99% of microbes on Earth exist in complex biofilm-like matrices, conferring crucial survival advantages to the microorganisms living within these structures⁶. More importantly, formation of these sessile domains is the cause of most persistent and chronic hospital-acquired infections⁷. Acinetobacter baumannii is one of the major human pathogens that is associated with global hospital-acquired infections and its virulence is largely attributed to quorum-sensing-mediated biofilm formation⁸. Quorum-quenching enzymes have been used successfully in disrupting quorum-mediated signal transduction by targeting a group of compounds known as N-acyl homoserine lactones (AHLs) that are produced by Gram-negative bacteria⁹. Several studies have also expanded upon the use of these enzymes to block bacterial pathogenesis through the reduction of virulence factor expression and cell numbers in biofilms^10,11. Unfortunately, there remains a lack of palpable demonstration of the effective use of quorum-quenching enzymes against biofilm formation by bacterial pathogens. There have been attempts to use quorum inhibitors (AHL analogues), instead of quorum-quenching enzymes, to disrupt A. baumannii biofilm formation¹². Although this method of using small molecules inhibitors is a valid approach, sustaining its bioavailability in translational uses can be a challenge. On the contrary, the use of catalytic quorum-quenching enzymes could circumvent the bioavailability issue as enzymes are more amenable towards immobilization on surfaces of biomedical devices for therapeutic effects.

Here, we describe an assessment of the effects of engineered quorum-quenching lactonases from Geobacillus kaustophilus (GKL)¹³ on bacterial biofilm formation, using crystal violet staining and confocal laser scanning microscopy (CLSM). This study is the first successful demonstration of biofilm disruption in a clinically relevant A. baumannii S1 strain using quorum-quenching enzymes. The methods described in this study are useful for assessing the efficacy of other quorum-quenching enzymes in subsequent therapeutic development efforts against pathogenic Gram-negative bacteria.

方案

1. Crystal Violet Quantitation of Biofilm Formation in A. baumannii S1

1. Grow a 5 ml culture of A. baumannii S1 in Lysogeny broth (LB) (tryptone 10 g/L, yeast extract 5 g/L) at 30 °C in a shaking incubator (220 rpm) for 16 hr.
2. Adjust the culture of A. baumannii S1 to a desired OD₆₀₀ of 0.8. Using a 96-well plate, inoculate the bacteria culture (1:100 dilution) into fresh LB containing 10 µl of purified GKL enzyme (40 mg/ml); the new culture's final volume is 100 µl.
3. Prepare a control culture as well; this will not contain any enzyme. Repeat similar conditions to yield the desired number of replicates.
4. Cover the plate with a lid and place it into a sealed 10 L plastic container. Incubate the plate at 30 °C for 3 hr before gently removing the media.
5. Add another 100 µl of fresh LB medium to the well and incubate the plate for 21 hr at 30 °C.
6. After the second period of incubation, gently remove all the media. Wash the planktonic bacteria cell with 200 µl sterile water. Ensure that there is only minimal disturbance to the cells during washing.
7. Add 100 µl of 1% crystal violet solution to each well and incubate for 15 min at RT. Remove the crystal violet solution by washing the well with 200 µl sterile water. Repeat the wash for two more times.
8. Add 100 µl of 33% acetic acid to each well and incubate for 15 min with gentle shaking; this will dissolve the dye.
9. Quantitate the amount of biofilm formed by measuring the absorbance of crystal violet at 600 nm. The amount of crystal violet is proportional to the amount of biofilm formed.

2. Confocal Laser Scanning Microscopy of A. baumannii S1 Biofilm

1. Grow a 5 ml culture of A. baumannii S1 in LB at 30 °C in a shaking incubator (220 rpm) for 16 hr.
2. Adjust the culture of A. baumannii S1 to a desired OD₆₀₀ of 0.8. Using a 35 mm glass-bottomed µ-Dish, inoculate the bacteria culture (1:100 dilution) into fresh LB containing 30 µl of purified GKL enzyme (40 mg/ml); the new culture's final volume is 1 ml.
3. Cover the µ-Dish with a lid and place it in a sealed 10 L plastic container. Incubate the µ-Dish at 30 °C for 3 hr before gently removing the media. Add 30 µl of purified GKL enzyme and fresh medium, bringing it to a total volume of 1 ml. Incubate for another 21 hr at 30 °C.
4. Repeat step 2.3 and incubate the µ-Dish for another 24 hr at 30 °C. Then, remove the media gently.
5. Add 500 µl of 5 µg/ml Alex Fluo 488-conjugated wheat germ agglutinin (WGA) dissolved in Hank's balanced salt solution (HBSS) to the µ-Dish and incubate at 37 °C for 30 min; this will stain the formed biofilm. Remove the staining solution and wash the µ-Dish with 2 ml of HBSS. Repeat the wash step one more time.
6. Add 500 µl of 3.7 % formaldehyde dissolved in HBSS and incubate at 37 °C for 30 min; this will fix the biofilm onto the µ-Dish. Wash the µ-Dish once with 2 ml of HBSS and then remove the solution completely. The µ-Dish fixed with biofilm can be stored in the dark at 4 °C prior to CLSM imaging.
7. For CLSM imaging and analysis, use 63 times magnification to generate 97 stacks per image with an interval of 0.21 µm per stack.

结果

In the crystal violet quantitation experiment, two quorum-quenching enzymes were used to demonstrate feasibility in disrupting biofilm formation: wild-type GKL and an improved GKL double mutant (E101G/R230C). Both enzymes have been shown to demonstrate lactonase activity against 3-hydroxy-decanoyl-L-homoserine lactone (3-OH-C₁₀-HSL), the major quorum molecule used by A. baumannii S1¹⁴. For valid assessment of biofilm disruption, their respective catalyticall...

讨论

In both sets of experiment, A. baumannii S1 was cultured in LB media without NaCl as a high salt concentration may reduce the amount of biofilm formed by the bacteria¹⁵. The presence of such artifact could underestimate the amount of biofilm formed, as well as the effects of quorum-quenching enzymes across different treatment conditions. The use of a catalytically inactive enzyme is important as a negative control to eliminate the possible effects of enzyme sequestration. Figure 1 sho...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
胰蛋白胨	BD	211705
酵母提取物	BD	212750
96 孔板	Costar	3596
结晶紫	Sigma-Aldrich	C6158
乙酸	实验室扫描	PLA00654X	注意：易燃
μ-Dish	Ibidi	80136
Alex Fluo 488 偶联 WGA	Invitrogen	W11261
汉克's 平衡盐溶液	Invitrogen	141475095
甲醛	Sigma-Aldrich	F8775	注意：腐蚀性
Synergy HT 微孔板检测仪	BioTek
1X-81 倒置荧光显微镜	奥林巴