Engineering soluble, high affinity T cell receptor domains for detection of staphylococcal and streptococcal exotoxin

Staphylococcus aureus and group A Streptococcus express a family of exotoxins including staphylococcal enterotoxins A, B, C (SEA, SEB, SEC), toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxins (SpeA, SpeC) that possess superantigenic properties, by virtue of which they stimulate a large fraction of an individual's T cells, leading to hyperinflammation and in some cases, organ failure and death. The molecular mechanism behind hyperinflammation has been attributed to the binding of superantigen (SAg) to both a T cell receptor (TCR) on a T cell, and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell, which leads to cross-linking of cells and excessive cytokine release from both cell types. Although SAgs bind with low affinity to the variable region of the beta chain (Vbeta) of the TCR, they are very potent toxins. These toxins have been incriminated in food poisoning, sepsis, toxic shock syndrome (TSS), infective endocarditis, and skin conditions like atopic dermatitis. For purposes of neutralizing or detecting SAg, soluble versions of several Vbeta domains against various SAgs have been engineered in the Kranz laboratory. Yeast display and directed evolution have allowed development of soluble, high-affinity Vbeta proteins some of which have also been successfully used in animal models of S. aureus infections. In this work, a high-affinity Vbeta receptor for Staphylococcal enterotoxin A (SEA) was generated, and the use of this and other engineered, high-affinity Vbeta proteins as specific and sensitive detecting agents was explored.;SEA is one among the collection of enterotoxins secreted by Staphylococcus aureus, which acts as a superantigen. It is also the most common enterotoxin recovered from food poisoning outbreaks in the United States. It is estimated that in most cases, the dose of SEA that causes the disease is of the order of a few micrograms per individual. In this study, I engineered a soluble form of a Vbeta that had high affinity toward SEA, for purposes of understanding its molecular interaction with SEA and also to develop specific, sensitive assays for detection of SEA.;In chapter 2, the process of engineering of human Vbeta22 protein for improving its affinity towards SEA is described. In this chapter, yeast display coupled with random mutagenesis and fluorescence-activated cell sorting (FACS) was used to select for stably expressing Vbeta22 mutants on yeast cell surface. Because the affinity of stabilized Vbeta22 mutants for SEA was low, these were then subjected to directed mutagenesis to select for mutants with enhanced affinity towards SEA. Incorporation of additional mutations by site-directed mutagenesis, yielded the high affinity mutant called "FL". Selected Vbeta22 mutants were expressed in E.coli and refolded in vitro, for assessing their binding properties. In collaboration with Dr. Eric Sundberg, the binding constants for interaction of wt Vbeta22 and high affinity "FL" protein with SEA were determined by surface plasmon resonance. FL protein was shown to bind SEA with a KD value of 4nM, which was a 25,000-fold improvement in affinity compared to the wild-type protein, which binds SEA with low affinity (KD ∼100microM). The SEA:Vbeta interface was centered around residues within the complementarity determining region 2 (CDR2) loop of the Vbeta. The engineered, high-affinity Vbeta22 protein was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. Finally, the high-affinity Vbeta22 mutant protein was used for development of a capture-ELISA based platform for specific detection of SEA.;In Chapter 3, a bead-based, two-color flow cytometry approach was used to develop a multiplex assay for simultaneous detection of staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins in a single sample. Vbeta domains that were engineered for binding with high-affinity to these toxins were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC) in singleplex assays. These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. The singleplex assays were combined to yield a multiplex assay that allowed reliable detection of the toxins in a single sample. In multiplex format, the sensitivity of detecting individual toxin was reduced due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected SEA, SEB and TSST-1 in supernatants derived from cultures of various strains of Staphylococcus aureus. Thus, these reagents and the flow cytometry-based platform can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes, or directly in clinical samples.