Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

Abstract

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d -/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ ‐mediated complement evasion to promote systemic infection in vertebrate hosts

“…Although some of these proteins are produced in abundance, most are expressed at low levels when cultured in vitro and/or during murine infection (Kenedy, Lenhart, & Akins, 2012), which have been hurdles in studying their roles in pathogenesis. Cultivating B. burgdorferi in specific conditions such as blood-supplemented growth media to induce the production of proteins of interest has been utilized to investigate the role of such proteins in vitro and in vivo (Kumar et al, 2015;Moriarty et al, 2012;Parveen & Leong, 2000;Zhi et al, 2015). We used human blood to treat spirochetes and observed induction of CspZ (Figure 1) as this host's blood has been used as a host-simulated cue to alter the expression of spirochetes genes (Tokarz et al, 2004).…”

Unraveling the host's immune response to infection: Seeing is believing

“…Notably, iNKT cell‐deficient animals have a significantly higher burden of bacteria and inflammation in the joint after infection . Humans are particularly susceptible to Lyme arthritis, thus Kumar et al . used IVM to further study the vascular transmigration of Borrelia burgdorferi into the joints using iNKT cell‐deficient mice.…”

“…used IVM to further study the vascular transmigration of Borrelia burgdorferi into the joints using iNKT cell‐deficient mice. They identified P66, a bacterial integrin adhesin and porin, to be required for vascular transmigration into the joint tissue by this spirochete …”

Tailored environments to study motile cells and pathogens

“…Borrelia , the causative agents of a number of human infections including Lyme disease are transmitted by ticks into the dermis, where they can migrate over long distances before entering into the blood stream to disseminate (Steere et al, ). Borrelia has not yet been imaged in the skin, but there are several studies investigating the bacteria in blood capillaries (Kumar et al, ; Moriarty et al, ; Norman et al, ). In vitro , Borrelia usually swim at high speed in straight lines and can undergo complex interactions with each other (Kudryashev et al, ).…”

“…(Steere et al, 2016). Borrelia has not yet been imaged in the skin, but there are several studies investigating the bacteria in blood capillaries (Kumar et al, 2015;Moriarty et al, 2012;Norman et al, 2008). In vitro, Borrelia usually swim at high speed in straight lines and can undergo complex interactions with each other .…”