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Title:Defining the mechanisms of microbial sensing among members of the toll-like receptor 2 sub-family
Author(s):Ranoa, Diana Rose
Director of Research:Tapping, Richard I.
Doctoral Committee Chair(s):Tapping, Richard I.
Doctoral Committee Member(s):Slauch, James M.; Kranz, David M.; Degnan, Patrick H.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Toll-like receptors (TLRs)
Toll-like receptor 1 (TLR1)
Toll-like receptor 2 (TLR2)
Toll-like receptor 10 (TLR10)
innate immune recognition
pathogen-associated molecular patterns (PAMPs)
host-pathogen interactions
lipopeptide recognition
ternary complex formation
Abstract:As pattern recognition receptors of the innate immune system, Toll-like receptors (TLRs) sense microbial components and mediate cell activation leading to protective inflammatory responses. TLRs are type-1 transmembrane receptors that are activated by forming dimers; an event driven by the coordinate binding of a cognate microbial ligand. Bacterial lipoproteins are the most potent microbial agonists for the TLR2 subfamily and this pattern recognition event induces cellular activation leading to host immune responses. TLR2 mediates cellular responses to triacylated and diacylated bacterial lipoproteins by forming heterodimers with either TLR1 or TLR6, respectively. Crystal structure analysis revealed that triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2 resulting in a stable ternary complex that drives intracellular signaling. However, the order of complex formation upon recognition of microbial components is poorly understood. The primary objectives of this dissertation are to define the sequence of events by which lipoproteins from bacteria are delivered to TLR1 and TLR2 leading to the formation of a stable TLR1/TLR2/lipoprotein ternary complex as well as to identify important amino acid residues in the TLR1 extracellular domain that are necessary for ligand recognition and/or formation of the dimer interface with TLR2. Chapter One of this thesis provides an overview of the field of innate immunity and a review of the latest developments in Toll-like receptor studies, with an emphasis on the members of the Toll-like receptor 2 subfamily. We describe in detail the extracellular domain of TLRs and their importance in ligand recognition. Chapter Two assesses the role of two lipid-binding serum molecules, lipopolysaccharide binding proten (LBP) and cluster of differentiation 14 (CD14), in the delivery of microbial components to TLRs 1 and 2. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by LBP and soluble CD14 (sCD14). However, the physical mechanism which underlies this increased sensitivity is not known. To address this, we have measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and the synthetic triacylated lipopeptide agonist Pam3CSK4. Importantly, addition of sub-stoichiometric amounts of either LBP or sCD14 significantly enhances formation of a TLR1/TLR2/Pam3CSK4 ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 is physically associated with the final ternary complex. Similar results were obtained using OspA, a naturally-occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies reveal that either LBP or sCD14 sensitize TLR-expressing cells to nanogram levels of either Pam3CSK4 lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins. Chapter Three describes the measurement of interactions between TLRs and lipopeptides to assess the possible competition between TLR1 and TLR10 for lipopeptides and the TLR2 co-receptor. Using microtiter plate assays, we have demonstrated that Pam3CSK4 induces the formation of the TLR1/TLR2 and TLR2/TLR10 heterodimeric complexes, and have further shown that ternary complex formation is ligand-specific and can be blocked by monoclonal antibodies against the TLRs. TLR2 was shown to prefer TLR1 as co-receptor compared to TLR10 for assembly of a final ternary complex with Pam3CSK4. To quantitatively measure the kinetics of the interaction, surface plasmon resonance experiments based on a two-component system were initially performed. However, two major problems encountered in this system were the aggregation of the small 1.5 kDa Pam3CSK4 ligand in solution, as well as the non-uniformity of binding conditions after each regeneration step. To address these issues, we purified a larger 12kDa lipoprotein (Lip12) expressed in E. coli and used the Octet system to obtain uniform and repeatable results. The calculated dissociation constant (KD) of Lip12 binding to Fc-tagged TLRs 1, 2, and 10 immobilized on protein A sensors were 7.45x10-7 M (chi2: 0.0193), 1.58x10-6 M (chi2: 0.02892), and 3.34x10-7 M (chi2: 0.07317), respectively, based on a 1:1 Langmuir binding model. Moreover, TLR4 did not show any binding to Lip12, and neutralizing mAbs against TLR2 and TLR1 prevented interaction with Lip12, suggesting that the binding events are real and specific. TLR1 showed the highest affinity for the agonist, followed by TLR10, and finally by TLR2. This, together with the reported CD14-Pam3CSK4 interaction (KD = 5.7x10-6M) suggest that CD14 preferentially delivers the lipopeptide to TLR1 first followed by the binding of TLR1-lipopeptide to TLR2. In cells that express both TLR1 and TLR10, a competition for the agonist could take place, and the receptor that has a higher expression level on the cell surface would most likely win. Measuring the KD in a three-component system, while a critical step, is outside the scope of current work until the issues of protein aggregation, handling lipoproteins in solution, and steric hindrance brought about by non-specific dimerization of Fc-tagged TLRs can be resolved. Addition of LBP and/or sCD14 to this system did not resolve the aggregation problem. Chapter Four addresses the important regions in the TLR1 extracellular domain (ECD) that are required for activation of TLR1/TLR2 in response to a variety of natural agonists. We have previously shown that the central leucine-rich repeat motifs (LRRs 9 through 12) of TLR1 and TLR 6 are critical for lipoprotein discrimination. TLR1/TLR2 and TLR2/TLR6 heterodimers also mediate responses to a wide variety of other acylated microbial ligands and we show here that similar to lipopeptides, LRRs 9-12 of TLR1 and TLR6 are also required for this sensing function. To further delineate the residues important for this function, random mutagenesis was used to create a library of TLR1 clones with various single amino acid substitutions within LRRs 9-12. Using this library, the epitope of GD2.F4, an inhibitory anti-TLR1 mAb, was mapped to amino acid residues located in the flexible loop of LRR11. Additionally, the amino acids F314, P315, Q316 and V339, within LRRs 11 and 12, were each found to be necessary for the sensing of triacylated lipopeptides by TLR1. More importantly, mutation of these same critical residues greatly inhibited cellular responses to other TLR1/TLR2 microbial agonists. These results demonstrate that regardless of the molecular structure of the agonist, the same critical residues of TLR1 are required for ternary complex formation with TLR2 to initiate cellular activation. These residues lie at the interface between TLR1 and TLR2 in the TLR1/TLR2/lipopeptide complex suggesting that the overall structure of the ternary complex is the same regardless of the activating microbial agonist. Chapter Five summarizes the important findings from this dissertation, highlighting the contributions and implications of this work to the TLR field. We also describe future experiments that are predicted to provide further insights on the structure and function relationship between TLRs and their agonists. A better understanding of TLR sensing of bacterial cell wall components may lead to improved therapeutic strategies for treating inflammatory diseases.
Issue Date:2014-05-30
URI:http://hdl.handle.net/2142/49652
Rights Information:Copyright 2014 Diana Rose Ranoa
Date Available in IDEALS:2014-05-30
2016-09-22
Date Deposited:2014-05


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