Optimisation of bioluminescent reporters for use with mycobacteria.
Optimisation of bioluminescent reporters for use with mycobacteria.
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DOI：
10.1371/journal.pone.0010777
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发表时间：
2010-05-24
期刊：
影响因子：
3.7
通讯作者：
Wiles S
中科院分区：
文献类型：
--
作者：
Andreu N;Zelmer A;Fletcher T;Elkington PT;Ward TH;Ripoll J;Parish T;Bancroft GJ;Schaible U;Robertson BD;Wiles S
结核分枝杆菌是结核病的病原体，在许多国家仍然是一个主要的公共卫生威胁。生物发光是一种多功能的报告技术，在体外和体内都有多种应用。体内生物发光成像（BLI）是其最突出的用途之一，它允许在活体动物体内非侵入性定位表达端粒酶的细胞。尽管发光报告基因在分枝杆菌中广泛使用，但所得发光菌株尚未完全应用于BLI。使用生物发光进行体内成像的主要障碍之一是实现足够高的报告蛋白表达水平以获得可以在外部检测到的信号。因此，作为将该技术应用于体内分枝杆菌感染研究的第一步，我们使用载体、启动子和密码子优化基因的组合优化了萤火虫、Gaussia和细菌芽孢杆菌酶在分枝杆菌中的使用。我们首次报道了完整细菌lux操纵子在结核分枝杆菌和结核分枝杆菌中的功能表达。因此允许自发光分枝杆菌的发展。我们证明，高斯荧光素酶是从细菌细胞分泌，这种分泌不需要一个信号序列。最后，我们证明，由表达萤火虫或细菌荧光素酶的重组分枝杆菌产生的信号可以在感染小鼠的肺部通过生物发光成像进行非侵入性检测。虽然还有很多工作要做，但萤火虫和细菌的双酶都可以在活小鼠中非侵入性地检测到，这一发现是使用这些报告基因研究M.结核病和其他分枝杆菌物种。此外，自发光分枝杆菌的发展对于高通量分枝杆菌药物筛选测定具有巨大的影响，目前使用LuxAB或萤火虫荧光素酶以破坏性方式进行高通量分枝杆菌药物筛选测定。
Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI. One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging. While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.
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影响因子：
9.4
作者：
ANDREW, PW;ROBERTS, IS
通讯作者：
ROBERTS, IS
影响因子：
4.1
作者：
Galluzzi, Lorenzo;Karp, Matti
通讯作者：
Karp, Matti
影响因子：
4.9
作者：
Arain, TM;Resconi, AE;Stover, CK
通讯作者：
Stover, CK
影响因子：
5.5
作者：
Dennehy, Maureen;Bourn, William;Williamson, Anna-Lise
通讯作者：
Williamson, Anna-Lise
DOI：
10.1099/13500872-141-8-1785
发表时间：
1995-08-01
期刊：
MICROBIOLOGY-UK
影响因子：
--
作者：
DELLAGOSTIN, OA;ESPOSITO, G;MCFADDEN, J
通讯作者：
MCFADDEN, J