Applying molecular and phenotypic screening assays to identify efficient quorum quenching lactonases.
Applying molecular and phenotypic screening assays to identify efficient quorum quenching lactonases.
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DOI：
10.1016/j.enzmictec.2022.110092
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发表时间：
2022-10
影响因子：
3.4
通讯作者：
Daude, David
中科院分区：
文献类型：
--
作者：
Billot, Raphael;Plener, Laure;Grizard, Damien;Elias, Mikael H.;Chabriere, Eric;Daude, David
群体感应（QS）是微生物利用细胞密度依赖性方式采取行为的分子通讯系统。能够水解信号分子酰基高丝氨酸内酯（阿勒）的内酯酶可以抵消革兰氏阴性菌中QS介导的毒力。因此，通过酶工程方法优化阿勒的内酯酶活性或特异性对于增加保护作用是非常有吸引力的。然而，仅开发了有限数量的筛选方法来处理和评估AHL降解酶库。在这里，开发了一系列的筛选程序，以确定改进的内酯酶使用两个以前报道的酶作为基准，即SsoPox和GcL。具体地，使用六种不同的阿勒并基于两种报告菌株进行分子筛选;即，紫色色杆菌CV 026和恶臭假单胞菌KS 35。此外，三个基于表型的筛选，旨在评估酶的能力，以淬灭特定的QS相关的行为，使用C。紫色假单胞菌、铜绿假单胞菌和哈维氏弧菌为致病型菌株。这些测定用于筛选小型文库，并允许鉴定各种改进的变体。为了证实这些变体是真实的“命中”，产生并纯化了其中的四种。发现它们对阿勒底物的动力学参数与起始酶相比增加了2-44.5倍。此外，通过测量它们在不同细菌系统中淬灭QS的能力，证实了它们增加的活性。这些新的检测方法将有助于筛选酶库，并为开发熟练的工程QS破坏酶铺平道路。
Quorum sensing (QS) is a molecular communication system used by microorganisms to adopt behaviors in a cell density-dependent manner. Lactonase enzymes, able to hydrolyze the signal molecules acyl-homoserine lactones (AHL) can counteract QS-mediated virulence in Gram-negative bacteria. Optimizing lactonases activity or specificity for AHL through enzyme engineering approaches is thus highly attractive to increase protective effect. However, only a limited number of screening methods have been developed to handle and evaluate AHL-degrading enzyme libraries. Here, a series of screening procedures were developed to identify improved lactonases using two previously reported enzymes as benchmarks, namely SsoPox and GcL. Specifically, molecular screenings using six different AHL and based on two reporter strains; i.e., Chromobacterium violaceum CV026 and Pseudomonas putida KS35, are reported. In addition, three phenotype-based screenings aiming to evaluate the ability of enzymes to quench a particular QS-related behavior are reported, using C. violaceum, Pseudomonas aeruginosa and Vibrio harveyi as pathogenic type strains. These assays were used to screen a small-sized library and allowed for the identification of various improved variants. To confirm that these variants were real “hits”, four of them were produced and purified. Their kinetic parameters against AHL substrates were found to be increased by 2–44.5 -fold as compared to the starting enzyme. Moreover, their increased activity was confirmed by measuring their ability to quench QS in different bacterial systems. These new assays will facilitate the screening of enzyme libraries and will pave the way for the development of proficient engineered QS-disrupting enzymes.
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影响因子：
5.8
作者：
Kang, Chang-Ho;Kim, YongGyeong;So, Jae-Seong
通讯作者：
So, Jae-Seong
DOI：
10.1099/00221287-143-12-3703
发表时间：
1997-12-01
期刊：
MICROBIOLOGY-UK
影响因子：
--
作者：
McClean, KH;Winson, MK;Williams, P
通讯作者：
Williams, P
影响因子：
5.2
作者：
Mahan, Kathleen;Martinmaki, Ryan;Elias, Mikael
通讯作者：
Elias, Mikael
DOI：
10.1107/s2053230x16011821
发表时间：
2016-09-01
影响因子：
0.9
作者：
Bergonzi, Celine;Schwab, Michael;Elias, Mikael
通讯作者：
Elias, Mikael
影响因子：
4.9
作者：
Fernandez, Lucia;Breidenstein, Elena B. M.;Hancock, Robert E. W.
通讯作者：
Hancock, Robert E. W.