An extensively validated whole-cell biosensor for specific, sensitive and high-throughput detection of antibacterial inhibitors targeting cell-wall biosynthesis.
An extensively validated whole-cell biosensor for specific, sensitive and high-throughput detection of antibacterial inhibitors targeting cell-wall biosynthesis.
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DOI：
10.1093/jac/dkac429
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发表时间：
2023-03-02
影响因子：
5.2
通讯作者：
O'Neill, Alex J.
中科院分区：
文献类型：
--
作者：
Galarion, Luiza H.;Mitchell, Jennifer K.;Randall, Christopher P.;O'Neill, Alex J.
全细胞生物传感器菌株是抗菌药物发现的强大工具，原则上可以识别作用于特定、高价值目标途径的抑制剂。虽然已经描述了多种用于检测细胞壁生物合成抑制剂（CWBI）的生物传感器，但这些菌株通常缺乏特异性和/或敏感性，并且在很大程度上没有作为主要筛选工具进行严格评估。在这里，我们描述了几种金黄色葡萄球菌 CWBI 生物传感器，并表明基于特异性和灵敏的生物传感器的 CWBI 发现是可以实现的。生物传感器包含 lacZ 报告基因与金黄色葡萄球菌启动子（PgltB、PilvD、PmurZ、PoppB、PORF2768、PsgtB）的融合，这些启动子在细胞壁生物合成抑制后受到上调。通过使用荧光或发光底物测量β-半乳糖苷酶的表达来检测生物传感器的诱导。所测试的六种生物传感器中的三种（基于 PgltB、PmurZ、PsgtB 的生物传感器）在 CWBI 存在的情况下表现出明显的 β-半乳糖苷酶表达特异性诱导。使用大量阳性和阴性对照化合物和条件突变体对其中之一 (PmurZ) 进行进一步验证，确定其对细胞壁生物合成的抑制有适当且独特的反应。使用这种生物传感器，我们建立、验证并部署了一种高通量测定法，该测定法从超过 9000 种天然产物提取物的筛选中鉴定出一种潜在的新型 CWBI。我们经过广泛验证的 PmurZ 生物传感器菌株可对 CWBI 进行特异性、灵敏的检测，非常适合高通量筛选；因此，它是抗菌药物发现的一个有价值的工具。
Whole-cell biosensor strains are powerful tools for antibacterial drug discovery, in principle allowing the identification of inhibitors acting on specific, high-value target pathways. Whilst a variety of biosensors have been described for detecting cell-wall biosynthesis inhibitors (CWBIs), these strains typically lack specificity and/or sensitivity, and have for the most part not been rigorously evaluated as primary screening tools. Here, we describe several Staphylococcus aureus CWBI biosensors and show that specific and sensitive biosensor-based discovery of CWBIs is achievable. Biosensors comprised lacZ reporter fusions with S. aureus promoters (PgltB, PilvD, PmurZ, PoppB, PORF2768, PsgtB) that are subject to up-regulation following inhibition of cell-wall biosynthesis. Induction of biosensors was detected by measuring expression of β-galactosidase using fluorogenic or luminogenic substrates. Three of the six biosensors tested (those based on PgltB, PmurZ, PsgtB) exhibited apparently specific induction of β-galactosidase expression in the presence of CWBIs. Further validation of one of these (PmurZ) using an extensive array of positive and negative control compounds and conditional mutants established that it responded appropriately and uniquely to inhibition of cell-wall biosynthesis. Using this biosensor, we established, validated and deployed a high-throughput assay that identified a potentially novel CWBI from a screen of >9000 natural product extracts. Our extensively validated PmurZ biosensor strain offers specific and sensitive detection of CWBIs, and is well-suited for high-throughput screening; it therefore represents a valuable tool for antibacterial drug discovery.
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DOI：
10.1111/j.1749-6632.1974.tb43277.x
发表时间：
1974-01-01
影响因子：
5.2
作者：
KAHAN, FM;KAHAN, JS;KROPP, H
通讯作者：
KROPP, H
DOI：
10.1073/pnas.2014810118
发表时间：
2021-01-19
影响因子：
11.1
作者：
Kearney KJ;Butler J;Posada OM;Wilson C;Heal S;Ali M;Hardy L;Ahnström J;Gailani D;Foster R;Hethershaw E;Longstaff C;Philippou H
通讯作者：
Philippou H
影响因子：
2.1
作者：
HAYES, MV;CURTIS, NAC;WARD, JB
通讯作者：
WARD, JB
DOI：
10.1039/c39850000254
发表时间：
1985-01-01
影响因子：
--
作者：
BARNA, JCJ;WILLIAMS, DH;WILLIAMSON, MP
通讯作者：
WILLIAMSON, MP
影响因子：
1.2
作者：
Figueroa, Dania M.;Wade, Heidi M.;Darling, Louise E. O.
通讯作者：
Darling, Louise E. O.