In Vivo Imaging of Meningococcal Disease Dynamics
Neisseria meningitidis is a human specific organism that causes severe sepsis and/or meningitis with high mortality. The disease scenario is rapid and much remains unknown about the disease process and host–pathogen interaction. In this chapter, we descri
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Introduction Luciferase enzymes, present in certain bacteria, marine crustaceans, fish, and insects, can generate light through the oxidation of an enzyme-specific substrate (commonly known as luciferin) in the presence of oxygen and energy (1). Photon emission can be detected by a light sensitive apparatus, such as a charge-coupled device (CCD) camera. A significant advantage of luciferases as optical indicators is the sensitivity and high signal intensity in live mammalian cells and tissues. Bioluminescent reporters have been applied intensively to monitor biological and disease processes in cell lines and live animal models (2–5). The luxCDABE operon, originating from the soil bacterium Photorhabdus luminescens, carries all the genes essential for luminescence production. luxAB encodes the alpha and beta subunits of the heterodimeric luciferase and luxCDE encodes the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction (6). The luxCDABE operon can be Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_10, © Springer Science+Business Media, LLC 2012
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efficiently expressed in Gram-negative bacteria and the encoded luciferase is functional at temperatures as high as 45°C. It is therefore ideally suited for real-time monitoring of microbial physiology and infectious disease in vivo (7). In this chapter, we describe a protocol for generating a bioluminescently labeled Neisseria meningitidis strain based on the luxCDABE operon. We also show how in vivo BLI revealed intriguing disease dynamics during meningococcal infection (8). Bioluminescent N. meningitidis strains combined with suitable animal disease models provide a potent tool for in vivo investigations of the pathogenesis of meningococcal infection, the role of pathogenic factors, as well as the efficacy of vaccine candidates.
2. Materials 2.1. Bacteria and Growth Conditions
1. N. meningitidis strain FAM20 is stored in GC medium containing 30% (v/v) glycerol at −80°C (see Note 1). 2. E. coli strain DH5A is stored in Luria Bertani (LB) medium with 30% (v/v) glycerol at −80°C. 3. Kellogg’s supplement (9): 40% (w/v) D-glucose, 0.5% (w/v) L-glutamine, 0.05% (w/v) ferric nitrate, and 0.01% (w/v) cocarboxylase. Sterilize by passage through a 0.2-Mm filter and store at 4°C. 4. GC agar plates: dissolve 36 g of GC agar powder (Acumedia) in 1 L of distilled water. Autoclave for 15 min at 121°C and 2.68 kg/cm2 and then cool to around 50°C, add 10 mL of Kellogg’s supplement before pouring into sterile plastic Petri dishes. 5. GC medium: dissolve 15 g of protease peptone (Oxoid), 1 g of soluble starch, 4 g of K2HPO4, 1 g of KH2PO4, and 5 g of NaCl in 1 L of distilled water. Autoclave and store at room temperature. 6. Luria Bertani (LB) medium: dissolve 25 g of LB broth powder (Acumedia) in 1 L of distilled water, autoclave, and store at room temperature. 7. LB agar plates: dissolve 37 g
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