Abstract
Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.
Highlights
Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever and Lyme borreliosis [1,2,3,4,5,6]
We were able to engineer infectious and non-infectious B. burgdorferi expressing a highly fluorescent green fluorescent protein (GFP) allele optimized for bacterial expression [20,31] (Fig. 1A); this allele is distinct from the egfp [21,23] and gfpmut1 [21,23] alleles that have been expressed in B. burgdorferi
98.7 2/+ 1.3% of the B. burgdorferi cultivated in the absence of gentamycin retained robust levels of GFP expression (Fig. 1B), indicating that the GFP-expressing plasmid was stably maintained in the context of the murine host
Summary
Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever and Lyme borreliosis [1,2,3,4,5,6]. Many clinically-important spirochetes cross the blood-brain barrier and exhibit an unusual form of motility that is predicted to permit efficient movement through dense extracellular matrix in host tissues [6,7,8,9]. Spirochetes of the Borrelia burgdorferi sensu lato species complex are the causative agents of Lyme borreliosis [1,10]. B. burgdorferi are transmitted to the skin of mammalian hosts through the bite of an infected tick. They enter the vascular circulation and disseminate hematogenously to multiple tissues by unknown mechanisms. Untreated Lyme borreliosis can result in arthritis, carditis and neurological complications
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