title: Invasion of human tissue ex vivo by Borrelia burgdorferi.
abstract: Borrelia burgdorferi sensu stricto is an etiological agent of Lyme disease. The lack of an adequate ex vivo system for human tissue infection is an obstacle to fully understanding the molecular mechanisms of invasion of tissue by B. burgdorferi and its adaptation within the human host. Here, we report on the development of such a system. We inoculated blocks of human tonsillar tissue with B. burgdorferi spirochetes, cultured them in a low-shear rotating wall vessel (RWV) bioreactor, and analyzed them using light and electron microscopy, nested polymerase chain reaction (PCR), and quantitative real-time PCR. Also, we evaluated the expression of the outer surface proteins (Osps) OspA and OspC by use of quantitative Western blotting. Light and electron microscopic analysis revealed multiple spirochetes localized extracellularly within the tissue, and their identity was confirmed by PCR. Quantification of spirochetes inside the RWV-cultured tonsillar tissue demonstrated that the number of B. burgdorferi exceeded the initial inoculum by an order of magnitude, indicating that spirochetes replicated in the tissue. Electron microscopic analysis showed that some spirochetes were arranged in cystic structures and that invading spirochetes differentially expressed surface proteins; both of these features have been described for infected tissues in vivo. The system we have developed can be used to study B. burgdorferi pathogenesis under controlled conditions ex vivo, in particular to explore the gene activation responsible for the adaptation of B. burgdorferi to human tissue that leads to Lyme disease.
authors.affiliation: Department of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. paul.duray@med.va.gov
authors.forename: Paul H
authors.initials: PH
authors.lastname: Duray
authors.forename: Shu-Rong
authors.initials: SR
authors.lastname: Yin
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ito
authors.forename: Ludmila
authors.initials: L
authors.lastname: Bezrukov
authors.forename: Cheri
authors.initials: C
authors.lastname: Cox
authors.forename: Myong-Soon
authors.initials: MS
authors.lastname: Cho
authors.forename: Wendy
authors.initials: W
authors.lastname: Fitzgerald
authors.forename: David
authors.initials: D
authors.lastname: Dorward
authors.forename: Joshua
authors.initials: J
authors.lastname: Zimmerberg
authors.forename: Leonid
authors.initials: L
authors.lastname: Margolis
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001428:Bacterial Vaccines; D001431:Bacteriological Techniques / Q000379:methods*; D025065:Borrelia burgdorferi / Q000502:physiology* / Q000648:ultrastructure; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008074:Lipoproteins / Q000378:metabolism; D014066:Palatine Tonsil / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D008074:Lipoproteins; C072045:OspA protein; C079616:OspC protein