The Borrelia burgdorferi Integrin Ligand P66 Affects Gene Expression by Human Cells in Culture

Abstract

Borrelia burgdorferi, an agent of Lyme disease, establishes persistent infection in immunocompetent animals and humans. Although the infection in humans can be cleared by antibiotic therapy, persistence in reservoir animals is necessary for the maintenance of the bacterium in the natural reservoir hostNtick vector infectious cycle. B. burgdorferi binds to ␤ 1 -and ␤ 3 -chain integrins, and the P66 outer membrane protein is responsible for at least some of the integrin binding activity of the spirochete. Because integrins are transmembrane, bidirectional signaling molecules, integrin binding may alter the nature of the host response to the bacteria. We used isogenic B. burgdorferi p66؉ and ⌬p66 strains to analyze the responses of cultured human cells to P66-integrin interaction during infection. Microarray results suggest that the response differs according to the cell type, infection time, and experimental conditions. Clusters of genes in functionally related categories that showed significant changes included proteins involved in cell-extracellular matrix interactions, actin dynamics, stress response, and immune responses. Integrin binding by P66 may therefore help B. burgdorferi establish infection by facilitating tissue invasion and modulating the activation of the immune system to other components of the bacteria, e.g., lipoproteins. These results provide insight into how B. burgdorferi is able to establish infection in immunocompetent hosts.

Integrin binding byBorrelia burgdorferi P66 facilitates dissemination but is not required for infectivity

“…Both leukocyte and B. burgdorferi transmigration across the endothelium likely occur at endothelial cell‐cell junctions, as B. burgdorferi has been observed in the intercellular junctions between endothelial cells (Szczepanski et al ., ; Moriarty et al ., ). The P66‐integrin interaction may function to initiate signalling cascades to facilitate transmigration across the endothelium, including VEGF/VPF, as previously reported (LaFrance et al ., ), which would represent a novel function for bacterial integrin ligands. Alternatively, B. burgdorferi may use the integrin interactions with P66 as traction in cooperation with the motility of the bacterium to drive through the endothelial barrier.…”

“…Human embryonic kidney cell line 293 (HEK293) stably expressing human integrin α v β 3 (293 + α v β 3 ) (LaFrance et al ., ) was cultured in Dulbecco's modified Eagle medium plus Ham's F12 nutrient mix (base medium; Gibco) with 5% heat‐inactivated fetal bovine serum (FBS) (Gibco) supplemented with 400 μg ml −1 of G418 (Gibco). Human microvascular endothelial cells (HMEC‐1) (Ades et al ., ) were cultured in MCDB 131 (base medium; Gibco) with 15% heat‐inactivated Hyclone FBS (Hyclone Laboratories), 1 μg ml −1 of hydrocortisone (Sigma‐Aldrich), 10 ng ml −1 of epidermal growth factor (Gibco) and 25 mM HEPES (Gibco).…”

“…The numbers of monocytes, neutrophils and other cells of the host defence system at the inoculation site did not differ in mice inoculated with Δp66 bacteria as opposed to the wild‐type (WT) parental strain (Ristow et al ., ), suggesting that the clearance of the Δp66 bacteria is mediated through host defences already in place, as opposed to defence cells that migrate to the site of infection. Microarray studies comparing endothelial cell responses to B. burgdorferi revealed an up‐regulation of the vascular endothelial growth factor/vascular permeability factor pathway in cells incubated with WT bacteria compared with those incubated with Δp66 bacteria (LaFrance et al ., ). This result suggested that one function of P66 might be to facilitate bacterial transmigration across endothelial barriers.…”

Characterization of Multiprotein Complexes of the Borrelia burgdorferi Outer Membrane Vesicles

“…Additionally, P66 has also been shown to modulate the expression of a specific set of genes in cultured human cells 53 . However, similar to other transport proteins, such as P13 54 , DipA 55 , BB0142 56 , MIP family proteins 57, 58 or oligopeptide permeases (Opp) complex 59, 60 , P66 is also suggested to be involved in solute transport across the borrelial membrane 61, 62 .…”

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

“…While the ability of P66 to bind ␤ 3 -chain integrins and to form pores has been well established (30,(32)(33)(34)(99)(100)(101), the relative contributions of the pore-forming and integrin-binding capabilities of P66 to spirochetal virulence are not clear. Although P66 is required for establishing an infection in mice, it is not essential for the growth of B. burgdorferi in dialysis membrane chambers (36).…”