Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host

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. Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme disease. They exhibit an unusual form of motility and can infect many different tissues; however, the mechanism by which they disseminate from the blood to target sites is unknown. Direct visualization of bacterial pathogens at the single cell level in living hosts is an important goal of microbiology, since this approach is likely to yield critical insight into disease processes. We engineered a fluorescent strain of Borrelia burgdorferi, a Lyme disease pathogen, and used conventional and spinning disk confocal intravital microscopy to directly visualize these bacteria in real time and 3D in living mice. We found that spirochete interaction with and dissemination out of the vasculature was a multi-stage process of unexpected complexity and that spirochete movement appeared to play an integral role in dissemination. To our knowledge, this is the first report of high resolution 3D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.