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Motility of Lyme Disease Spirochetes in Fluids as Viscous as the Extracellular Matrix Robert B. Kimsey and Andrew Spielman

From the Department of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts

The apparent scarcity of the Lyme disease spirochete (Borrelia burgdorferi) in the blood of infected hosts [1] seems paradoxical if hematophagous Ixodes ticks are to serve as vectors. B. burgdorferi seem to concentrate in internal viscera where ticks cannot feed and in the skin near the mouthparts of feeding ticks [2]. Spirochetes are also present in skin lesions [3]. Although these spirochetes may disseminate passively in circulating fluids, active movement would be required for migration through skin. A viscoelastic matrix of glycoproteins, comprising various forms of collagen and glycosaminoglycans, fills the extracellular space of that tissue [4]. Although spirochetes and flagellated bacteria swim through such thin fluids as blood, they move most rapidly in thicker solutions that contain small amounts of such compounds as methylcellulose [5-7]. Gel-like nonvascular fluids, such as those in the joints of the long bones, and the aqueous and vitreous humors of the eye may also be invaded [8]. Thus, Lyme disease spirochetes might be most motile in fluids that approach the physical properties of the dermal ground substance. Indeed, intradermal dissemination of the Lyme disease spirochete would account for the migratory nature of the erythema migrans rash that accompanies the early phase of infection. The peculiar abundance of Lyme disease spirochetes in fixed tissues and their apparent absence in blood may derive from an ability to migrate most effectively through such highly viscoelastic fluids as those in the extracellular matrix of the skin. Accordingly, we identified characteristic properties of the medium that might limit the ability of these organisms to loco-

Received 16 October 1989; revised 20 March 1990. Financial support: AI-19693 (National Institutes of Health); DAMD-17-87C-7110 (US Army Research and Development Command). Reprints or correspondence: Dr. Andrew Spielman, Department of Tropical Public Health, 665 Huntington Ave., Boston, MA 02115. The Journal of Infectious Diseases 1990;162:1205-1208 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6205-0036$01.00

mote, seeking to correlate rate of progression with viscoelasticity. In particular, we recorded motility in an array of solutions of glycosaminoglycans and methylcellulose.

Materials and Methods B. burgdoiferi were obtained from a laboratory culture (JDl Harvard strain) maintained in BSK II medium at 33°C. Spirochetes were maintained in a constant state of high-productivity growth by frequent high-volume passage. Virtually all spirochetes harvested from these cultures and used in the following experiments moved actively. All of the following were obtained from Sigma Chemical (St. Louis). Glycosaminoglycans (GAGs) mixed into media for measuring spirochete motility included chondroitin sulfate (CS) derived from bovine tracheae (C-8528) and hyaluronic acids (HA) derived from human umbilical cord (H-1751), bovine trachea (H40150), and rooster comb (H-7262). Also, 2% methylcellulose (MC; M-0512; viscosity of a 2 % solution at 25°C, /"\J4000 centipoise [cpl). In addition, a mixture of 0.5 % CS from bovine trachea in 1% rooster comb HA was prepared. All solutions were diluted in PBS. Spirochetes were introduced into these media by diluting with BSK II medium in which these organisms were grown. Six dilutions of each solution were prepared in a geometric series from 1% to 0.0625 % MC or GAG. Samples were stirred for 5 s with a vortex mixer and centrifuged at 1000 g for 5 s to remove bubbles. Aliquots of each dilution were pipetted onto separate glass slides and sealed under a coverslip with petroleum jelly. The rate of movement of spirochetes was measured microscopically [6, 9]. Rapid movements were recorded on videotape at x320 using a video recorder and monitor connected to a video camera mounted on a microscope fitted for darkfield illumination. Measurements of the time and distance traveled by spirochetes at room temperature were made using interval tracings on plastic film overlaying the monitor when the tape was played back. Slow movements were recorded and measured without video recording. Measurements began 1-1.5 h after samples were sealed and continued for 1 h thereafter. The microscope field was shifted systematically to ensure that no spirochete was measured twice. Measurements were recorded as spirochetes moved on the surface of the slide and while suspended

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When properties of extracellular fluids that might regulate the ability of the Lyme disease spirochete to locomote were investigated, the rate of progression correlated with viscoelasticity. Such spirochetes flexed and rotated but did not progress in relatively nonviscous fluids and migrated increasingly rapidly as the viscous characteristics ofthe medium increased. The viscoelastic properties of various kinds of hyaluronic acid resembled those of a methylcellulose standard. The maximum velocity that spirochetes achieved in such solutions related directly to viscoelasticity rather than to chemical composition. Spirochetes remained motile during 3 h ofobservation despite tOO-fold dilution of the standard nutrient medium. The immobility of Lyme disease spirochetes in media less viscous in character than fixed tissue suggests dissemination via the intercellular ground substance of skin.

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Results First we determined how viscous properties of a fluid affect Lyme disease spirochete motility by adding various quantities of MC to spirochetes suspended in BSK II medium. As viscoelasticity of the medium increased from 1.3 to 205 cp, mean velocity of spirochetes increased from 1.7 to 34.9 p.m/s (figure lA). The rate of increase diminished as viscosity approached 200 cpo Thus, Lyme disease spirochetes moved more rapidly as the viscoelasticity increased. The viscoelasticity of dissolved GAGs was then compared with that of Me. Various HAs were chosen to represent the class of GAGs that innately lack a core protein, and CS was chosen to represent those in which the core is lost during preparation. CS was much less viscoelastic than were any of the HAs (figure IB). HA from human umbilical cord had the greatest viscoelasticity and that from bovine trachea the least; rooster comb HA was intermediate. At greatest concentration, human umbilical cord HA did not pour, rooster comb HA poured slowly, and bovine trachea HA poured readily. Thus, the viscoelastic properties of various kinds ofHA resembled those of the MC standard. In diluent alone, spirochetes tended to progress more effectively when in contact with a glass surface than when in suspension. In all except the most viscoelastic solutions of rooster comb HA, however, $pirochetes suspended within the medium and not in contact with a surface generally moved about as rapidly as those in contact with the surface of the slide (figure 2A). In the most viscous solutions, the rate of progressive movement of surface-associated spirochetes exceeded that of suspended spirochetes. In the experiments that follow, therefore, all observations were based on suspended spirochetes. We then compared the motility of Lyme disease spirochetes suspended in various HAs prepared in a range of dilutions in BSK II. Movement in rooster comb HA corresponded closely to that in the MC standard (figure 2A). Motility in bovine trachea HA only slightly exceeded that in BSK II diluent, even at the highest concentration tested (1 %) (figure 2B). Human umbilical cord HA, however, greatly enhanced

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Figure 1. A, Rate of movement of Lyme disease spirochetes in various concentrations of methylcellulose ( rv 4000 cp at 2 %). Each point represents mean of 10 fastest moving of at least 100 spirochetes. Bars indicate 1 SD. B, Viscoelasticity of various concentrations of methylcellulose and hyaluronic acid (HA) from rooster comb, bo.., vine trachea, and human umbilical cord.

spirochete motility (figure 2B). The spirochetes moved most rapidly when the concentration reached 0.5% (100 cp); movement slowed at 1.0% (1050 cp). Rate of movement increased in rooster comb HA throughout the range of concentrations used (1 %, 205 cp). We concluded that the velocity of spirochetes in these media varied with the concentration of HA or MC and related to viscoelasticity rather than to the chemical properties of the agent used to regulate viscosity. We then determined whether the rate of movement of suspended Lyme disease spirochetes varied with the nutrient content of the media. Rooster comb HA was diluted in BSK II or in PBS (1 % solution). In nondiluted BSK II, mean motility was f'V34 p.m/s, compared with 32 p.m/s in diluted medium. The spirochetes remained constantly motile during the 3 h ofobservation, even in diluted medium. Thus the nutrient content of the medium did not affect movement of spirochetes.

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in media. At least 100 spirochetes were observed at each dilution of MC or GAG. To compare differing activity rates among batches of spirochetes, the velocity of spirochetes in undiluted BSK II medium was measured with each new viscoelastic agent. Following a standard procedure, we calculated the average and standard deviation of the velocity of the 10 most rapidly moving spirochetes in each dilution [6, 9-11]. We analyzed the flow properties of media by means of CannonManning semi-microcalibrated viscometers (sizes 150, 300, 400, 500) using procedures set out in ASTM standard D-445; measurements are given in centipoise units of viscosity. This convention incorporates both Newtonian and non-Newtonian flow characteristics of solutions, including viscosity, elasticity, and shear [7]. The mean maximum rate of progression of spirochetes was thereby plotted as a function of viscoelasticity for each agent.

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Figure ·2. A, Rate of movement of suspended spirochetes compared with that of spirochetes in contact with a surface in various concentrations of rooster comb hyaluronic acid. B, Rate of movement of spirochetes suspended in different concentrations of human umbilical cord and bovine trachea hyaluronic acid (HA).

Discussion The ability ofthe Lyme disease spirochete to swim depended on the viscoelasticity of the suspended fluid, and motility was greatest at rv1200 cpo Greater viscous properties inhibited swimming. Translational movements of other bacteria, including certain spirochetes, also correlate with viscoelasticity [6, 9]. Flagellated bacteria move most rapidly at rv2 cp but become immobilized at 60 cp [6, 11]. Escherichia coli and Campylobacter pyloridis swim fastest at 8 and 10 cp, respectively [9]. Such spirochetes as Spirillum gracile and Spirochaeta halophila move optimally at 5-8 cp and become immobile at 1000 cp [111. Leptospira species, however, move best in more viscous media; the optimum approaches 300 cp [10]. Adaptation to viscous media seems to characterize these spirochetes; B. burgdorferi and Leptospira species appear far more specialized for viscous environments than are other organisms.

Helical morphology of bacteria facilitates dispersal in viscoelastic environments [5, 7, 9, 11], and diverse groups ofbacteria resemble Borrelia in this regard. They generally exploit such environments, and many require such a niche [9, 12, 13]. Cristispira, for example, inhabits only the highly viscoelastic matrix of the crystalline style of mollusks, and commensal bacteria, such as Campylobacter and Treponema species, specialize in the mucus of the vertebrate gut. The ability to penetrate mucus-covered tissue surfaces gives pathogenic Leptospira and Treponema species the competence to invade and establish infection in the vertebrate body [10]. Perhaps the Lyme disease spirochete resides in similarly viscous sites within its hosts. GAGs, mainly HA and CSs, exhibit exceptional viscoelasticity; they comprise the ground substance that fills the space between collagen fibers [4]. Connective tissues consist of such a matrix, particularly in the dermis; this matrix forms a continuous nexus throughout the body. The ground substance of the dermis consists principally of HA and dermatan sulfate and to a lesser extent, ofchondroitin-4-sulfate and chondroitin6-sulfate [4]. HA, a long-chain polymer of disaccharide subunits, traps 1000 to 10,000 times its volume of water [14]. Thus, the dermis provides a continuous system of canals filled with GAGs, and our observations suggest that the Lyme disease spirochete may effectively migrate through such sites. Feeding vector ticks would deliver the agent directly to the dermis and imbibe them from this tissue. The peculiar ability of this spirochete to migrate rapidly through GAGs may assist its escape from the inflammatory response of vertebrate hosts. Indeed, seronegative Lyme disease in human hosts is not uncommon [15]. Because phagocytic tissue macrophages are less motile, they could not capture the spirochete and initiate the immune cascade by presenting antigen to T helper cells. Thus, these fluid-filled connective tissues may shelter spirochetes, potentially a reservoir of chronic infection that may later recrudesce. Although Lyme disease spirochetes rarely can be demonstrated in blood [3], the soft-tick and louse-borne relapsing fevers include prominent spirochetemias, a difference that corresponds to the contrasting modes of feeding of the vectors. Thus, the rapid-feeding vectors ingest the corresponding Borrelia organism from blood, and the slow-feeding hard ticks appear to ingest the Lyme disease spirochete from fixed tissues. Only a slow-feeding vector could effectively drain spirochetes from the dermis and serve as a focus around which they might concentrate [2]. The translational movements of spirochetes depend on physical properties of the substrate. Interestingly, Leptospira organisms swim readily in solutions of MC that form a loose molecular meshwork but not in the spherular colloid formed by ficoll [7]. Native collagen fibers, similar to molecules of MC, form a semirigid meshwork [4]. The intracellular matrix of the dermis is mesh-like, suggesting particular dermal adaptations.

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Percent Hyaluronic Acid

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The Lyme disease spirochete cannot move along its longitudinal axis unless it is suspended in a medium with viscous properties that approach those of fixed tissue. The relative immobility of Lyme disease spirochetes in less viscoelastic media suggests active dissemination via the intercellular ground substance of skin.

References

6. Schneider WR, Doetsch RN. Effect of viscosity on bacterial motility. J Bacteriol 1974;117:696-701 7. Berg HC, Turner L. Movement of microorganisms in viscous environments. Nature 1979;245:249-351 8. Johnson YE, Duray PH, Steere AC, Kashgarian M, Buza J, Malawista SE, Askenase PW. Lyme arthritis: spirochetes found in synovial microangiopathic lesions. Am J Pathol 1985;118:26-34 9. Hazeil SL, Lee A, Brady L, Hennessy W. Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. J Infect Dis 1986;153:658-663 10. Kaiser GE, Doetsch RN. Enhanced translational motion of Leptospira in viscous environments. Nature 1975;255:656-657 11. Greenberg EP, Canale-Parola E. Motility of flagellated bacteria in viscous environments. J Bacteriol 1977;132:356-358 12. Tall BD, Nauman RK. Scanning electron microscopy of Cristispira in Chesapeake Bay oysters. Appl Environ MicrobioI1981;42:336-343 13. Harwood CS, Canale-Parola E. Ecology of spirochetes. Ann Rev Microbiol 1984;38:161-192 14. Sharon N. Complex carbohydrates. Their chemistry, biosynthesis and functions. New York: Addison-Wesley, 1975 15. Dattwyler RI, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golighty MG. Seronegative Lyme disease. Dissociation of specific T- and Blymphocyte responses to Borrelia burgdorferi. N Engl J Med 1988; 319:1441-1446

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1. Benach JL, Bosler EM, Hanrahan JP, Coleman JL, Habicht GS, Bast TF, Cameron DJ, Zieglar JL, Barbour AG, Burgdorfer W, Edelman R. Spirochetes isolated from the blood of two patients with Lyme disease. N Engl J Med 1983;308:740-742 2. Nakayama Y, Spielman A. Ingestion of Lyme disease spirochetes by ticks feeding on infected hosts. J Infect Dis 1989;160:166-167 3. Steer AC, Grodzicki RL, Craft JE, Shrestha M, Kornblat AN, Malawista SE. Recovery of Lyme disease spirochetes from patients. Yale J BioI Med 1984;57:557-560 4. Mier PD, Cotton DWK. The molecular biology of the skin. Oxford: Blackwell, 1976 5. Goldstein SF, Charon NW. Motility of the spirochete Leptospira. Cell MotH Cytoskeleton 1988;9:101-110

lID 1990;162 (November)

Motility of Lyme disease spirochetes in fluids as viscous as the extracellular matrix.

When properties of extracellular fluids that might regulate the ability of the Lyme disease spirochete to locomote were investigated, the rate of prog...
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