PATHOGENESIS MICROBIAL Microbial Pathogenesis 41 (2006) 43–47 Short communication Detection of Borrelia burgdorferi gene expression during mammalian infection using transcriptional fusions that produce green fluorescent protein Jennifer C. Miller 1 , Kate von Lackum 2 , Michael E. Woodman, Brian Stevenson à Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, KY 40536-0298, USA Received 10 February 2006; received in revised form 5 April 2006; accepted 7 April 2006 Available online 24 May 2006 Abstract A novel, infectious Borrelia burgdorferi that expresses green fluorescent protein (GFP) was developed to examine the utility of this marker protein to label live bacteria during infection processes. Use of a borrelial erpAB promoter to direct gfp transcription supported previous indications that B. burgdorferi expresses erp genes during chronic mammalian infection and during acquisition by feeding ticks. Live bacteria fluoresced and were seen to be located extracellularly in infected mice and within midguts of infected ticks. These results indicate that transcriptional fusions between B. burgdorferi promoters and gfp can be useful tools to examine spirochete gene expression in vivo. r 2006 Elsevier Ltd. All rights reserved. Keywords: Borrelia burgdorferi; Spirochete; Gene fusion; Lyme disease 1. Introduction Lyme disease is a significant zoonosis affecting humans and domestic animals in many parts of the US, Europe, and other regions of the world. The causative agent, the spirochetal bacterium Borrelia burgdorferi, persists in nature through infectious cycles between many species of vertebrates and Ixodes spp. ticks. Newly hatched larval ticks acquire infections through ingestion of blood from infected vertebrate hosts. B. burgdorferi colonizes the larval midgut, and persists there through the molt to the nymph stage. Bacteria are then transmitted through the saliva when the nymph feeds on the next vertebrate host. Should that host be a human, the resultant infection may manifest in a variety of symptoms, including skin rashes, arthritis, meningitis, optic neuritis, facial nerve palsy and atrioven- tricular nodal block. B. burgdorferi can persistently infect immunocompetent humans and other mammals for many years [1]. All characterized isolates of B. burgdorferi naturally maintain multiple members of a highly conserved family of genetic elements, the cp32s [2]. Each cp32 carries a mono- or bicistronic erp locus, such that an individual bacterium may encode a dozen or more unique Erp proteins. Several of these outer membrane lipoproteins have been identified as binding host complement factor H, and are thereby hypothesized to protect B. burgdorferi against complement- mediated killing during mammalian infection [3]. Immuno- fluorescence analyses (IFA) indicated that essentially 100% of B. burgdorferi express every tested Erp protein both during transmission between infected ticks and naı¨ ve mammals and between infected mammals and naı¨ve ticks [4,5]. As bacteria are exposed to host blood at both those stages of the infectious cycle, these experimental results strengthen the hypothesized protective function of Erp proteins. Reverse transcription-PCR (RT-PCR) indicated that erp genes are also expressed by B. burgdorferi during long-term infection of mammals [6,7]. However, B. burgdorferi are exceedingly difficult to observe visually in infected mammalian tissues, so we have not previously ARTICLE IN PRESS www.elsevier.com/locate/micpath 0882-4010/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2006.04.004 à Corresponding author. Tel.: +1 859 257 9358; fax: +1 859 257 8994. E-mail address: brian.stevenson@uky.edu (B. Stevenson). 1 Present address: Department of Pathology, University of Utah, 30 North 1900 East, Salt Lake City, UT 84132-2501, USA. 2 Present address: Department of Oral Biology, University of Florida, PO Box 100424, Gainesville, FL 32610-424, USA.