Flentie Kelly N, Qi Min, Gammon Seth T, Razia Yasmin, Lui Felix, Marpegan Luciano, Manglik Aashish, Piwnica-Worms David, McKinney Jeffrey S
Department of Molecular Microbiology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Mol Imaging. 2008 Sep-Oct;7(5):222-33.
Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.
鼠伤寒沙门氏菌是人类肠胃炎的常见病因，在动物体内也定位于肿瘤组织。侵入特定的真核细胞是沙门氏菌与宿主组织相互作用的关键机制。胃肠道细胞侵袭的早期阶段由沙门氏菌III型分泌系统介导，该系统由三磷酸腺苷酶invC提供动力。本研究的目的是表征invC对侵入不同真核细胞（传统上用作肠道上皮或肿瘤模型）动力学的依赖性。因此，开发了一种非破坏性实时检测方法，以使用含有染色体整合luxCDABE基因的lux + 沙门氏菌来报告真核细胞侵袭动力学。使用lux + 沙门氏菌进行的基于生物发光的侵袭检测显示接种剂量反应相关性，区分有侵袭能力和无侵袭能力的沙门氏菌，并根据诱导侵袭基因表达的环境条件区分相对沙门氏菌侵袭性。在标准庆大霉素保护试验中，lux + 沙门氏菌的生物发光与内化细菌的菌落形成单位的回收率相关，并且可以通过生物发光显微镜观察到。此外，该检测可以实时区分有侵袭能力和无侵袭能力的细菌，而与庆大霉素处理无关。生物发光报告了沙门氏菌对不同真核细胞系的侵袭，包括用于恶性肿瘤动物模型的肿瘤性黑色素瘤、结肠腺癌和胶质瘤细胞系。在每种情况下，沙门氏菌对真核细胞的侵袭都依赖于invC。
