Tuesday, January 27, 2009
Watch videos of the Lyme disease spirochete escaping from the bloodstream of live mice!
Most pathogenic microbes that cause systemic infections, regardless of their route of host entry, migrate to the circulatory system, which facilitates their spread throughout the body. These invasive microbes, which include the Lyme disease spirochete B. burgdorferi, eventually exit the bloodstream and penetrate into various organs of the host. Last June in the online journal PLoS Pathogens, a Canadian research group presented some fascinating microscopic video footage of Borrelia burgdorferi traveling within and escaping from the bloodstream of live mice. We may like to think that the unique shape of the spirochete allows it to simply drill through the vessel wall, but the videos suggest that escape from the bloodstream is a little more complex.
Because spirochetes are too thin to observe by light microscopy, Moriarty and colleagues made B. burgdorferi fluoresce by transforming the spirochete with a gfp (green fluorescent protein) plasmid. To prepare the animals, they lifted the skin of anesthetized mice for observation of the underlying dermal microvasculature by fluorescence intravital microscopy (IVM), which allows visualization of cellular events in a living animal. They next injected the fluorescent spirochetes into the bloodstream of the mice, and they examined dermal postcapillary venules under the microscope as the spirochetes traveled through the field of view within the vessels.
The black-and-white video reveals several types of interactions between the spirochetes and vessel wall. The bar graph displayed below the video indicates the proportion of each type of interaction observed. Almost 90% of the contacts are transient, lasting for less than a second. About 10% of the interactions involved crawling or dragging of the spirochete along the vessel wall for up to 20 seconds. As you can see from the bar graph, these short-term interactions, although common, rarely lead to escape of spirochetes from the bloodstream. Perhaps the spirochetes crawl along the wall probing for an escape route from the vessel. When their search fails, as it usually does, they detach and float (or swim) away and try again elsewhere along the vessel wall. Occasionally, a spirochete will remain stuck to the vessel wall for many minutes. One such spirochete can be seen in the video, near the center of the screen. More careful observation of stationary spirochetes in the bloodstream of several mice revealed at least one end deeply embedded with the vessel wall, usually between endothelial cells. It is unclear whether these stationary adhesions are a necessary prelude to exit of the spirochete of the vessel as consistent outward movement of embedded spirochetes was never observed during the observation period, which lasted up to 45 minutes.
The next two videos capture spirochetes in the process of escaping from the bloodstream. The endothelium was stained by injecting the bloodstream with red fluorescent antibody to PECAM-1, a protein found within endothelial junctions. The first video shows how difficult it is for B. burgdorferi to traverse the wall of the venule. The spirochete appears to be stuck as it moves back and forth (reciprocal translation) across the vessel wall for several minutes trying to free itself. The second video shows a spirochete successfully dislodging itself and fleeing from the venule. The average escape time was 10.8 minutes (N = 11 spirochetes). The authors could not clearly determine whether the spirochetes escaped between or through endothelial cells.
Here's the model illustrating the steps in the escape of B. burgdorferi from the bloodstream. The spirochete first contacts and crawls (drag) across the inside surface of the vessel wall. It then crosses the vessel wall end-first. After a long period of back-and-forth motion (reciprocal translation), the spirochete finally escapes into the tissue. It is unknown whether stationary adhesion is necessary for escape.
Moriarty et al. repeated the experiments with B. burgdorferi rendered noninfectious by long-term passage in culture. They found minimal interaction of noninfectious spirochetes with the vessel wall, and not a single spirochete could be found escaping from the bloodstream. This result indicates that specific Borrelia surface molecules that are missing on noninfectious B. burgdorferi mediate interaction with and escape from the bloodstream. What are these B. burgdorferi surface molecules, and which host molecules do they contact in the blood vessel? Past in vitro experiments with cultured mammalian cells by several research groups have revealed a few candidates for such bacterial and host factors. The authors described the roles of these candidates in transient, dragging, and stationary adhesions in live mice in a follow-up study, which I will write about in my next post.
Tara J. Moriarty, M. Ursula Norman, Pina Colarusso, Troy Bankhead, Paul Kubes, George Chaconas (2008). Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host. PLoS Pathogens, 4 (6) DOI: 10.1371/journal.ppat.1000090