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Detection of Bioluminescent Borrelia burgdorferi from In Vitro Cultivation and During Murine Infection

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1690)

Abstract

Borrelia burgdorferi, etiologic agent of Lyme disease, is the leading tick-borne disease in the United States with approximately 300,000 cases diagnosed annually. Disease occurs in stages beginning localized infection at the site of a tick bite and progresses to disseminated infection when antibiotic treatment is not administered in a timely manner. A multi-systemic infection develops following dissemination to numerous immunoprotective tissues, such as the heart, bladder, and joints, resulting in late Lyme disease. B. burgdorferi undergoes dynamic genetic regulation throughout mammalian infection and defining the exact role of virulence genes at distinct stages of disease is challenging. The murine model allows for the characterization of the pathogenic function of genes in B. burgdorferi, but traditional end point studies limit the ability to gather data throughout an infection study and greatly increase the required number of mice. Molecular genetic techniques to evaluate and quantitate B. burgdorferi infection are laborious and costly. To partly circumvent these issues, a codon optimized firefly luciferase, under the control of a constitutive borrelial promoter, was introduced into B. burgdorferi enabling the characterization of mutant or modified strains under in vitro growth conditions and throughout murine infection. The detection of bioluminescent B. burgdorferi is highly sensitive and allows for the repeated real-time quantitative evaluation of borrelial load during murine infection. Furthermore, bioluminescence has also been utilized to evaluate alteration in tissue localization and tissue-specific gene expression of B. burgdorferi. In this chapter, we describe the generation of bioluminescent borrelial strains along with methods for in vitro, in vivo, and ex vivo B. burgdorferi studies.

Key words

Borrelia burgdorferi Spirochete Pathogen Bioluminescence Luciferase In vivo imaging Ex vivo imaging Infection Pathogenicity 

Notes

Acknowledgments

The previously published images modified for this chapter were supported by Public Health Service grants R01-AI058086 (to J.T.S.) and R21-AI101740-01 (to J.A.H.) from the National Institute of Allergy and Infectious Diseases. We also acknowledge Michael Norgard and Jon Blevins for the B. burgdorferi codon optimized luc gene. We also extend our gratitude to Jeffrey Cirillo, Geoffery Kapler, and Raquel Sitcheran for generously sharing equipment and resources necessary to develop these methods for B. burgdorferi. We wish to thank Kevin Francis, Will Hauser, and Brad Taylor at Perkin Elmer for their technical support and advice.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Microbial Pathogenesis and ImmunologyCollege of Medicine, Texas A&M Health Science CenterBryanUSA

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