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.

Featured paper

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

Sunday, January 18, 2009

The origin of syphilis: a phylogenetic approach

K.N. Harper, P.S. Ocampo, B.M. Steiner, R.W. George, M.S. Silverman, S. Bolotin, A. Pillay, N.J. Saunders, and G.J. Armelagos. (2008). On the origin of the treponematoses: A phylogenetic approach. PLoS Neglected Tropical Diseases 2(1):e148.

The first recorded outbreak of syphilis occurred in Europe in 1495, a few years after Columbus sailed the ocean blue. Was syphilis a New World disease newly introduced into Europe by Columbus and his crew, or was it an Old World disease that simply was not noticed until 1495? A study from Harper and colleagues published last January described a molecular genetic analysis that may have yielded important clues hidden within the genetic material of Treponema pallidum.

The authors first examined the evolutionary relationships among Treponema pallidum strains from subspecies pertenue, endemicum, and pallidum, which are responsible for the diseases yaws, bejel, and syphilis, respectively. The small collection of strains or their DNA was obtained from different patients throughout the past century. The tree illustrated below (Figure 3 of Harper et al.) was constructed from the alignment of 70 SNPs (single nucleotide polymorphisms) and 12 indels (insertions/deletions). The branching pattern indicates that pertenue emerged the earliest. Subspecies endemicum later emerged from pertenue, and pallidum, the agent of syphilis, arose most recently.
The authors also obtained scrapings from yaws skin lesions on two aboriginal children living deep in the rainforests of Guyana. Since these children were members of a population that had been living for generations with minimal contact with the rest of the world, these pertenue strains may be closely related to those present in the Americas before the European explorers arrived. Unfortunately, the samples collected by the authors had degraded extensively by the time the DNA was extracted for analysis. Consequently, the two Guyanan strains could not be included in the phylogenetic analysis shown above; only regions encompassing 17 of the 70 SNPs could be sequenced from the degraded DNA. Nevertheless, they went ahead and aligned the 17 nucleotides with those from the strains used to construct the phylogenetic tree. The alignment revealed that among the nonveneral strains (pertenue and endemicum), only the Guyanan strains had as many as 4 nucleotides that were identical to those of the pallidum strains.

The world map illustrated below (Figure 4 of Harper et al.) depicts the path of sequence changes in the 4 SNPs among the Treponema strains. The dots mark the geographic source of each strain used in the analysis. The red and green colors demark areas of endemicity of the nonvenereal diseases yaws and bejel, respectively, around the year 1900. The map shows that T. pallidum first appeared as pertenue in the Old World and gave rise to the endemicum subspecies, which migrated with humans to the Middle East and Europe. The Old World pertenue or endemicum strain then eventually gave rise to the New World pertenue strain as humans crossed the Bering Land Bridge and spread throughout the Americas. The sequence identity of the Guyanan strains with the pallidum strains at all 4 positions is consistent with the New World strain being introduced back into the Old World as a progenitor of today's syphilis-causing pallidum strains, which are now found worldwide. Clinical evidence also supports the New World model: the nonvenereal skin lesions in the Guyanan yaws patients resembled syphilis chancres rather than the typical skin lesions found with yaws.I do not believe that the results presented in the paper support the New World origin of syphilis. As Harper et al. state in the Discussion of the paper, the close evolutionary relationship of the South American pertenue strains with the pallidum (syphilis) strains is based on a mere four nucleotides. Still, the authors concluded that pallidum arose from a descendant of the New World pertenue that was brought to Europe by Columbus. However, the results do not rule out the possibility that pallidum and New World pertenue strains evolved independently from a common ancestor, such as the "unknown" strain illustrated in the map. The New World strains would need to be included in the phylogenetic tree to distinguish the two possibilities. It was unfortunate that the entire set of 70 SNPs and 12 indels could not be examined in the Guyanan strains.

Sunday, January 11, 2009

Chronic Lyme disease in mice?

E. Hodzic, S. Feng, K. Holden, K.J. Freet, and S.W. Barthold. (2008). Persistence of Borrelia burgdorferi following antibiotic treatment in mice. Infection and Immunity 52(5):1728-1736.

Controversy surrounds the management of those Lyme disease patients who continue to experience symptoms following treatment with the recommended course of antibiotics. These persisting symptoms, which include fatigue, sleep disturbances, and concentration difficulties, can be debilitating. The central question that underlies the controversy is whether the symptoms result from Borrelia spirochetes that survive treatment. If so, further treatment with oral or intravenous antibiotics, sometimes lasting for many months or even years, may be warranted, as Lyme disease advocates insist. However, the standard treatment guidelines do not recommend antibiotic treatment regimens lasting longer than one month. Supporters of the guidelines contend that post-treatment symptoms are not due to active Borrelia infection and that further treatment with antibiotics are not supported by clinical studies. Critics of the guidelines disagree with the interpretation of the clinical studies and cite cell culture and animal studies that suggest survival of Borrelia following antibiotic treatment. Insurance companies cite the guidelines in refusing to pay for costly long-term antibiotic treatment.

Early in 2008, Hodzic and colleagues at UC Davis published a study that examined the fate of Borrelia burgdorferi in infected mice treated for one month with ceftriaxone, an antibiotic commonly used to treat Lyme disease. The investigators succeeded in visualizing spirochetes in the tissues of a few mice. A typical example is presented in the image below (Figure 1 of the Hodzic et al. paper). Panel B shows a tissue section of a joint from a mouse that was treated with ceftriaxone initiated 4 months following infection with B. burgdorferi. The tissue was examined one month after antibiotic treatment was completed. Immunohistochemical staining clearly revealed a solitary spirochete (arrow). Panel A shows a joint from an infected mouse that was sham treated with saline. As expected, spirochetes were observed in tissues from most of the control mice, with up to four appearing in a section.

One could argue that the spirochetes observed in the ceftriaxone-treated mice were simply dead microbial carcasses awaiting removal. This interpretation appeared to be supported by the marked decrease in B. burgdorferi DNA copy number in mice tissues with time, as measured by quantitative PCR. However, ticks that fed on these mice were able to acquire and transmit the spirochetes to uninfected SCID mice, indicating that the spirochetes remained infectious despite ceftriaxone treatment. Yet all attempts to culture the bacteria from the treated mice, the ticks that fed on the mice, and even the SCID mice that the ticks fed on failed, even though B. burgdorferi was detected in the mouse tissues and ticks by PCR. In contrast, B. burgdorferi was successfully cultured from all control (saline treated) mice and the ticks that fed on the mice. These results suggest that B. burgdorferi that remained in treated mice were alive and infectious (and transmissible) but were impaired in their ability to replicate.

The most important question for those suffering from post-treatment symptoms is whether the spirochetes that survive antibiotic treatment cause clinical symptoms; the infectiousness of the spirochetes is less relevant. The authors microscopically examined the joints and hearts of the SCID mice for signs of inflammation. SCID mice are especially susceptible to developing severe inflammation when infected with B. burgdorferi. Nevertheless, inflammation was not detected in the SCID mice that acquired the disabled (yet infectious) spirochetes from ticks that previously fed on antibiotic-treated mice. This result does not rule out the possibility that disabled spirochetes contribute to post-treatment symptoms in humans by a microscopically undetectable mechanism. If residual spirochetes do indeed elicit clinical symptoms, then elimination of the spirochetes would be desired. Additional treatment with ceftriaxone may not be the best choice since it targets the cell walls of actively replicating bacteria. The mouse model developed by the UC Davis group will allow the investigators to test different treatment approaches for elimination of these persisting spirochetes.