Liu Mingyu, Blinn Christina, McLeod Sarah M, Wiseman John W, Newman Joseph V, Fisher Stewart L, Walkup Grant K
Biology Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, Massachusetts, United States of America.
RAD-Transgenics, Discovery Sciences, AstraZeneca R&D Mölndal, Mölndal, Sweden.
PLoS One. 2014 Mar 4;9(3):e90382. doi: 10.1371/journal.pone.0090382. eCollection 2014.
Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.
在动物感染模型中测量细菌载量是细菌致病机制研究和治疗药物研究的关键组成部分。传统的体内细菌载量定量方法需要频繁宰杀动物并对从感染部位回收的菌落形成单位（CFU）进行计数。为了解决这些问题，研究人员开发了多种表达荧光素酶的细菌报告菌株，以便在活体动物中进行细菌检测。迄今为止，所有此类基于荧光素酶的细菌报告基因均为细胞相关形式。分泌荧光素酶的重组细菌的产生可能具有从感染部位本身和远程采样（如体液和血浆）报告CFU的优势。为此，我们对致病性大肠杆菌（E. coli）菌株ATCC25922进行了基因改造，使其分泌海洋桡足类高斯伪镖水蚤荧光素酶（Gluc），并评估了Gluc作为小鼠组织笼感染中细菌载量的原位和异位报告基因的用途。表达Gluc的大肠杆菌在感染的组织笼模型中展示了细菌的体内成像。此外，在感染的18天内，从组织笼液（TCF）中回收的分泌型Gluc活性与细菌CFU相关。重要的是，一旦达到高细菌载量，在血浆样本中也可以检测到分泌型Gluc，并且它可以作为已建立的组织笼感染的异位指标。我们已经证明，来自海洋真核生物的Gluc可以在体内由致病性大肠杆菌稳定表达和分泌，从而为持续细菌感染的纵向评估提供一种简便的工具。
