Molecular Mechanisms Involved in Vascular Interactions of the Lyme Disease Pathogen in a Living Hostby M. Ursula Norman, Tara J. Moriarty, Ashley R. Dresser, Brandie Millen, Paul Kubes, George Chaconas

PLoS Pathog

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Year
2008
DOI
10.1371/journal.ppat.1000169
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Molecular Biology / Immunology / Microbiology / Parasitology / Virology / Genetics

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Molecular Mechanisms Involved in Vascular Interactions of the Lyme Disease Pathogen in a Living Host

M. Ursula Norman1.¤, Tara J. Moriarty2., Ashley R. Dresser2, Brandie Millen1, Paul Kubes1, George

Chaconas2* 1Department of Physiology & Biophysics, University of Calgary, Calgary, Alberta, Canada, 2Departments of Biochemistry & Molecular Biology and Microbiology &

Infectious Diseases, University of Calgary, Calgary, Alberta, Canada

Abstract

Hematogenous dissemination is important for infection by many bacterial pathogens, but is poorly understood because of the inability to directly observe this process in living hosts at the single cell level. All disseminating pathogens must tether to the host endothelium despite significant shear forces caused by blood flow. However, the molecules that mediate tethering interactions have not been identified for any bacterial pathogen except E. coli, which tethers to host cells via a specialized pillus structure that is not found in many pathogens. Furthermore, the mechanisms underlying tethering have never been examined in living hosts. We recently engineered a fluorescent strain of Borrelia burgdorferi, the Lyme disease pathogen, and visualized its dissemination from the microvasculature of living mice using intravital microscopy. We found that dissemination was a multistage process that included tethering, dragging, stationary adhesion and extravasation. In the study described here, we used quantitative real-time intravital microscopy to investigate the mechanistic features of the vascular interaction stage of B. burgdorferi dissemination. We found that tethering and dragging interactions were mechanistically distinct from stationary adhesion, and constituted the rate-limiting initiation step of microvascular interactions. Surprisingly, initiation was mediated by host Fn and GAGs, and the Fn- and GAG-interacting B. burgdorferi protein BBK32. Initiation was also strongly inhibited by the low molecular weight clinical heparin dalteparin. These findings indicate that the initiation of spirochete microvascular interactions is dependent on host ligands known to interact in vitro with numerous other bacterial pathogens. This conclusion raises the intriguing possibility that fibronectin and GAG interactions might be a general feature of hematogenous dissemination by other pathogens.

Citation: Norman MU, Moriarty TJ, Dresser AR, Millen B, Kubes P, et al. (2008) Molecular Mechanisms Involved in Vascular Interactions of the Lyme Disease

Pathogen in a Living Host. PLoS Pathog 4(10): e1000169. doi:10.1371/journal.ppat.1000169

Editor: Jenifer Coburn, Medical College of Wisconsin, United States of America

Received May 21, 2008; Accepted September 8, 2008; Published October 3, 2008

Copyright:  2008 Norman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to P.K. and G.C. (MOP-53086), and by a CIHR group grant.

P.K. and G.C. are Scientists of the Alberta Heritage Foundation for Medical Research (AHFMR), and Canada Research Chairs in, respectively, Leukocyte Recruitment in Inflammatory Disease, and Molecular Biology of Lyme Disease. M.U.N. and T.J.M. were each supported by postdoctoral fellowships from CIHR and AHFMR, and

M.U.N is an Australian NHMRC CJ Martin Fellow (284394).

Competing Interests: The authors have declared that no competing interests exist. * E-mail: chaconas@ucalgary.ca . These authors contributed equally to this work. ¤ Current address: Centre for Inflammatory Diseases, Department of Medicine, Monash University, Victoria, Australia

Introduction

Hematogenous dissemination of pathogenic organisms is an important feature of disease progression. However, dissemination is poorly understood, in large part because of the difficulty in studying this process directly in living organisms under the shear stress conditions that characterize the host vasculature. One such disseminating pathogen is the spirochete Borrelia burgdorferi, a primarily extracellular bacterium causing Lyme disease, also referred to as Lyme borreliosis [1].

Pathogenic spirochetes cause a number of emerging and reemerging diseases, including syphilis, leptospirosis, relapsing fever and Lyme disease [2–5]. B. burgdorferi is transmitted to the dermis of vertebrate hosts during the blood meal of Ixodes ticks, and subsequently disseminates to other tissues and organs during the hematogenous phase of infection [1]. B. burgdorferi and other spirochetes interact with endothelial cells under static conditions in vitro [6–8]. However, until recently, spirochete-vascular interactions have never been directly examined in the host itself, or under the fluid shear forces that are present at dissemination sites [9].

To facilitate direct study of hematogenous dissemination we recently generated a fluorescent infectious strain of B. burgdorferi, and used intravital microscopy (IVM) to directly visualize its interaction with and extravasation from the microvasculature of living murine hosts (as summarized in Fig. 1A–C) [9]. IVM is a powerful tool for studying the dissemination and transmigration of tumor and immune cells in living hosts, but it is only recently that this technique has begun to be applied to the study of hostpathogen interactions [10,11].

The results of our recent study indicated that B. burgdorferi dissemination from the host microvasculature in vivo is a progressive, multi-stage process consisting of several successive steps: transient and dragging interactions (collectively referred to as short-term interactions), followed by stationary adhesion and extravasation. Short-term interactions constitute the majority of spirochete-endothelial associations (89% and 10% for transient

PLoS Pathogens | www.plospathogens.org 1 October 2008 | Volume 4 | Issue 10 | e1000169 and dragging interactions, respectively), take less than one second (transient interactions) or 3–20s (dragging interactions) to travel 100 mm along the vessel wall, and occur primarily on the surface of endothelial cells and not at endothelial junctions [9]. Transient interactions are characterized by a tethering-type attachmentdetachment cycle of association in which part of the spirochete adheres briefly to the endothelium before being displaced by blood flow, whereas dragging spirochetes adhere along much of the length of the bacterium, and creep more slowly along the vessel wall [9]. In contrast, stationary adhesions (1% of interactions) do not move along the vessel wall, occur chiefly, but not exclusively, at endothelial junctions, and entail a more intimate association with the endothelium than short-term interactions [9]. Finally, spirochete extravasation (,0.12% of interactions) also occurs primarily, but not exclusively, at endothelial junctions, and is a triphasic process consisting of a rapid, end-first initial penetration of the endothelium, followed by a prolonged period of reciprocating movement, and ending with a rapid exit phase in which the bacterium bursts out of the vessel and migrates rapidly into the surrounding tissue [9].