Upon transmission by a vector tick bite, Lyme disease spirochetes, primarily B. burgdorferi (Bb) in the US, establish a local skin infection, then disseminate to multiple tissues. Chronic infection by Bb is often associated with arthritis. Our laboratories have identified and/or characterized many Bb cell- or extracellular matrix (ECM)- binding adhesins using multiple approaches, overcoming the challenges of defining their roles in Bb biology. Our approaches include analyses of biochemical activities and generation of targeted mutants selectively defective for a single adhesive activity and analysis of the mutants in multiple murine infection models. To gain detailed mechanistic insight into interactions that may occur during Bb dissemination in vivo, following intravenous inoculation we used intravital microscopy to characterize vascular attachment and transmigration in skin and joint-proximal tissue. These studies revealed that adhesins BBK32 and VlsE account for virtually all of the transient Bb-endothelium binding occurring minutes after inoculation, termed “Meeting” interactions. A distinct set of adhesins, DbpB/A, OspC, and P66, mediate contacts required for invasion into extravascular joint space after 24 hours (hr), termed “Transmigrating” interactions. Using isogenic strain sets that have acquired or lost specific adhesive activities in multiple short-term and long-term infection models we showed roles for five of the six Meeting or Transmigrating adhesins in short-term tissue localization and/or long-term colonization in other murine infection models. While Transmigrating adhesins do not promote “Meeting” interactions, our discovery of enhanced adhesive capacity of the endothelium as infection progresses has helped clarify why different adhesins have roles at different stages of infection. Within hr, “Endothelial Activation” permits BBK32- and VlsE-independent (“Greeting”) interactions. After ~24 hr “Endothelial Potentiation” occurs, reflected by the ability of the joint vasculature to support Bb transmigration. Although both OspC and P66 function as Transmigrating adhesins, only P66, an integrin-binding adhesin that alters transcription in cultured endothelial cells, is also Potentiating, i.e., required to promote the rapid transmigration of a second Bb strain. Activation is mimicked by exogenous treatment of mice with several cytokines produced by infected mice, but potentiation is seen only with TNF-α, MCP-1 or IL-10. These findings reveal previously unrecognized steps that are critical for Bb spread and provide a means to distinguish roles for each adhesin in distinct infection stages: Meeting, Greeting, Potentiating, Transmigrating and Colonizing. In Aim 1 we will identify known adhesins that facilitate Greeting interactions; to better prioritize our adhesin list, we propose a genome-wide screen that may also identify novel adhesins. In Aim 2 we will clarify the roles of known (and, if applicable, novel) adhesins in endothelial Pot...