INFECTION AND IMMUNITY, Jan. 2008, p. 298–307 Vol. 76, No. 1 0019-9567/08/$08.00+0 doi:10.1128/IAI.00866-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved. Global Transcriptome Analysis of Borrelia burgdorferi during Association with Human Neuroglial Cells  † Jill A. Livengood, Virginia L. Schmit, and Robert D. Gilmore, Jr.* National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Division of Vector-Borne Infectious Diseases, Bacterial Diseases Branch, Centers for Disease Control and Prevention, Fort Collins, Colorado 80521 Received 26 June 2007/Returned for modification 4 October 2007/Accepted 24 October 2007 As adherence and entry of a pathogen into a host cell are key components to an infection, identifying the molecular mechanisms responsible for cellular association will provide a better understanding of a microbe’s pathogenesis. We previously established an in vitro model for Borrelia burgdorferi infection of human neuroglial cells. To expand on our earlier study, we performed B. burgdorferi whole-genome expression analysis following a 20-hour infection of human neuroglial cells to identify borrelial genes that were differentially regulated during host-cell association compared with cultured Borrelia in cell-free medium. This study identifies several regulated genes, the products of which may be important mediators of cellular pathogenesis. Lyme disease is a complex illness caused by infection with the tick-borne spirochetal bacterium Borrelia burgdorferi and can present with multiple manifestations, such as arthritis, car- ditis, and neurological syndromes. B. burgdorferi adapts to disparate environments when trans- mitted between ticks and mammals. In naturally and experi- mentally infected murine hosts, B. burgdorferi has been found in a variety of tissues, including heart, bladder, joints, and ears (1, 2, 6, 58). In vitro, B. burgdorferi has been observed to adhere to endothelial cells, chondrocytes, synovial cells, peripheral blood fibrocytes, skin fibroblasts, tick cells, macrophages, and neuroglial cells of several species (12, 23, 27, 34, 35, 41, 42, 62). This ability to inhabit diverse environments implies that B. burgdorferi has the capability to adapt its physiology and mem- brane structure in response to its particular tissue location within arthropod and mammalian hosts. For example, investi- gators have demonstrated that B. burgdorferi inversely regu- lates the expression of outer surface proteins OspC and OspA as the spirochete migrates from the mid-gut of the tick to a mammal (45, 59). B. burgdorferi gene expression is orches- trated by many factors, including pH (7), temperature (46, 51, 59, 61), and host immune response (36–38). However, little is known about B. burgdorferi gene expression during spirochetal colonization of different tissues or cell types. Furthering our knowledge of B. burgdorferi differential gene expression during cellular infection would increase our understanding of the pathogen’s adaptive mechanisms responsible for dissemination and colonization to host cells and identify novel targets for development of new treatments and diagnostics. Subsequent to human infection with B. burgdorferi, neuro- logical syndromes occur in roughly 15% of the untreated pa- tients diagnosed with Lyme disease (60). The current case definition for diagnosis of acute neurological manifestations of Lyme disease includes meningitis, radiculopathy, cranial neu- ropathy, mononeuropathy complex and, rarely, encephalo- myelitis (67). Mechanisms by which B. burgdorferi affects the nervous system are not known, but association with and/or invasion of neural cells by this bacterium could be a basis for the clinical manifestations seen in neuroborreliosis. The nonhuman primate (NHP) is currently the most widely accepted model used to study late Lyme disease and neurobor- reliosis (49). B. burgdorferi-infected NHPs show both central and peripheral nervous system involvement. In NHPs infected with B. burgdorferi, researchers have detected spirochetes in central nervous system (CNS) tissue by PCR and, moreover, spirochetes have also been visualized by histopathological staining (5, 17, 48, 49, 55, 56). Although studies of Lyme disease in NHPs have yielded valuable data, in vitro cellular association studies have the ability to provide insights into the molecular mechanisms utilized by B. burgdorferi that contrib- ute to human neuroborreliosis. We have previously shown that an infectious strain of B. burgdorferi invades human endothelial cells and human neural cells in vitro (40). To obtain a more comprehensive under- standing of the borrelial mechanisms involved during cellular association, including attachment and invasion, we used mi- croarray analysis to determine the expression profile of B. burgdorferi upon encountering human neuroglial cells. Identi- fying genes that are differentially regulated during association with human host cells may provide clues to understanding borrelial mechanisms of neuropathogenesis and present poten- tial targets for development of novel diagnostics, prevention tools, and/or treatments for Lyme disease. MATERIALS AND METHODS Bacterial strains and growth conditions. B. burgdorferi strain B31 A3 is a clonal, low-passage infectious strain (16). Frozen stocks of all B. burgdorferi strains were maintained in 60% glycerol at -70°C. All bacterial strains were grown in liquid Barbour-Stoenner-Kelly (BSK-II) complete medium at 35°C with 5% CO 2 . * Corresponding author. Mailing address: Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, 3150 Rampart Road, CSU Foothills Campus, Fort Collins, CO 80521. Phone: (970) 221-6405. Fax: (970) 221-6476. E-mail: rbg9@cdc.gov. † Supplemental material for this article may be found at http://iai .asm.org/.  Published ahead of print on 5 November 2007. 298 on October 30, 2015 by guest http://iai.asm.org/ Downloaded from