Biology subjects

Skare, J. T.

Publications and source records attributed to Skare, J. T..

5 recordsLinked to original sources

bioRxiv2025

Identification of proteins exhibiting in vitro RNA chaperone activity through gradient profiling in the Lyme disease spirochete, Borrelia burgdorferi

RNA-binding proteins (RBPs) play key roles in regulating gene expression in bacteria. However, relatively few RBPs have been discovered and characterized in Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease. We utilized gradient profiling to identify putative RBPs that co-sediment with small RNAs (sRNAs) and nascent mRNAs. We employed in vitro and in vivo assays to characterize the RNA chaperone activities of several proteins we identified in the gradient profiling. The previously hypothetical proteins BB0749, BB0713 and BB0796, as well as the chemotaxis-related protein CheY2 and the flagella-associated protein FlgV displayed RNA annealing and/or strand displacement activity. Moreover, in vivo Co-IP assays demonstrated BB0749 binds RNA in B. burgdorferi.

molecular biology↗

bioRxiv2024

The dual activity of BBK32: Implications for simultaneous inhibition of borrelial-specific antibody-dependent complement activation and fibronectin binding

Complement inhibition is exploited by extracellular pathogens to combat clearance. Borreliella burgdorferi, the causative agent of Lyme disease, harnesses complement evasion techniques to establish and maintain infection in mammalian hosts. B. burgdorferi encodes bbk32, a surface lipoprotein that binds extracellular matrix (ECM) components, specifically glycosoaminoglycans (GAGs) and fibronectin (Fn) within its amino terminus. In addition to its ECM-binding functions, the carboxy terminal half of BBK32 binds to the C1r protease and prevents complement activation. This classical complement inhibitory activity protects B. burgdorferi from complement-mediated killing following the addition of normal human serum. Herein we demonstrate that full length BBK32 binds both Fn and C1 concurrently, indicating that binding of these macromolecules do not sterically hinder their simultaneous interaction. Given the link of antibody dependence to the classical pathway, we tested how the presence of BBK32 would protect B. burgdorferi from antibody mediated, complement dependent killing. The presence of BBK32 provided protection against borrelial-specific antibody binding and concomitant complement activation in vitro. We also demonstrated, using both fluorescence microscopy and flow cytometry, that the presence of BBK32 was required for reduced C4 deposition on the surface of borrelial cells. This work demonstrates the potential for BBK32 to simultaneously bind to both C1r and Fn and contributes to the broader understanding of the ability of B. burgdorferi to evade antibody-dependent complement-mediated killing. We contend that these observations ostensibly provide B. burgdorferi with coincident dissemination and immune evasion activities needed for optimal survival during infection. AUTHOR SUMMARYLyme disease, caused by Borreliella burgdorferi and other related species, is the most common arthropod-borne infection in the United States. As an extracellular pathogen, B. burgdorferi is exposed to the complement system--a soluble proteolytic cascade that clears invaders. Complement is defined by three pathways known as the alternative, lectin, and classical. The classical complement cascade is activated by the binding of antibodies to a foreign or damaged cell. For B. burgdorferi, the BBK32 surface protein is known to mute the classical pathway by binding and inhibiting the initiating protease C1r. However, no studies have addressed how BBK32 protects infectious B. burgdorferi from borrelial-specific antibody binding and clearance. Here we show that native BBK32 protects infectious B. burgdorferi from antibody-dependent, complement mediated killing. Given BBK32s other activity--namely its known adherence to fibronectin--we were interested to test if BBK32 could bind the C1 complex, which contains C1r, together with fibronectin. Our results suggest that these complex macromolecules can bind BBK32 simultaneously. These observations suggest that the dual activity of BBK32, namely fibronectin binding and C1r inhibition, are not mutually exclusive and contribute to B. burgdorferis ability to establish infection and evade antibody-based host clearance, respectively.

microbiology↗

bioRxiv2023

Conformational dynamics of C1r inhibitor proteins from Lyme disease and relapsing fever spirochetes

Borrelial pathogens are vector-borne etiological agents of Lyme disease, relapsing fever, and Borrelia miyamotoi disease. These spirochetes each encode several surface-localized lipoproteins that bind to components of the human complement system. BBK32 is an example of a borrelial lipoprotein that protects the Lyme disease spirochete from complement-mediated attack. The complement inhibitory activity of BBK32 arises from an alpha helical C-terminal domain that interacts directly with the initiating protease of the classical pathway, C1r. Borrelia miyamotoi spirochetes encode BBK32 orthologs termed FbpA and FbpB, and these proteins also inhibit C1r, albeit via distinct recognition mechanisms. The C1r-inhibitory activities of a third ortholog termed FbpC, which is found exclusively in relapsing fever spirochetes, remains unknown. Here we report the crystal structure of the C-terminal domain of B. hermsii FbpC to a limiting resolution of 1.5 [A]. Surface plasmon resonance studies and assays of complement function demonstrate that FbpC retains potent BBK32-like anti-complement activities. Based on the structure of FbpC, we hypothesized that conformational dynamics of the complement inhibitory domains of borrelial C1r inhibitors may differ. To test this, we utilized the crystal structures of the C-terminal domains of BBK32, FbpA, FbpB, and FbpC to carry out 1 {micro}s molecular dynamics simulations, which revealed borrelial C1r inhibitors adopt energetically favored open and closed states defined by two functionally critical regions. This study advances our understanding of how protein dynamics contribute to the function of bacterial immune evasion proteins and reveals a surprising plasticity in the structures of borrelial C1r inhibitors.

microbiology↗

bioRxiv2021

A structural basis for inhibition of the complement initiator protease C1r by Lyme disease spirochetes

Complement evasion is a hallmark of extracellular microbial pathogens such as Borreliella burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed co-crystallography and size exclusion chromatography-coupled small angle x-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. The studies reported here, for the first time, provide a structural basis for classical pathway-specific inhibition by a human pathogen.

microbiology↗

bioRxiv2020

The Intergenic Small Non-Coding RNA ittA is Required for Optimal Infectivity and Tissue Tropism in Borrelia burgdorferi

Post-transcriptional regulation via small regulatory RNAs (sRNAs) has been implicated in diverse regulatory processes in bacteria, including virulence. One class of sRNAs, termed trans-acting sRNAs, can affect the stability and/or the translational efficiency of regulated transcripts. In this study, we utilized a collaborative approach that employed data from infection with the Borrelia burgdorferi Tn library, coupled with Tn-seq, together with borrelial sRNA and total RNA transcriptomes, to identify an intergenic trans-acting sRNA, which we designate here as ittA for infectivity-associated and tissue-tropic sRNA locus A. The genetic inactivation of ittA resulted in a significant attenuation in infectivity, with decreased spirochetal load in ear, heart, skin and joint tissues. In addition, the ittA mutant did not disseminate to peripheral skin sites or heart tissue, suggesting a role for ittA in regulating a tissue-tropic response. RNA-Seq analysis determined that 19 transcripts were differentially expressed in the ittA mutant relative to its genetic parent, including vraA, bba66, ospD and oms28 (bba74). Subsequent proteomic analyses also showed a significant decrease of OspD and Oms28 (BBA74) proteins. To our knowledge this is the first documented intergenic sRNA that alters the infectivity potential of B. burgdorferi. AUTHOR SUMMARYLyme disease is a tick-borne infection mediated by the spirochetal bacterium, Borrelia burgdorferi, that is responsible for greater than 300,000 infections in the United States per year. As such, additional knowledge regarding how this pathogen modulates its regulatory armamentarium is needed to understand how B. burgdorferi establishes and maintains infection. The identification and characterization of small, non-coding RNA molecules in living systems, designated as sRNAs, has recalibrated how we view post-transcriptional regulation. Recently, over 1,000 sRNAs were identified in B. burgdorferi. Despite the identification of these sRNAs, we do not understand how they affect infectivity or B. burgdorferi pathogenesis related outcomes. Here, we characterize the ittA B. burgdorferi sRNA and show that it is essential for optimal infection using murine experimental infection as our readout. We also track the effect of this sRNA on the transcriptional and proteomic profile as the first step in providing mechanistic insight into how this important sRNA mediates its regulatory effect.

microbiology↗