Engineering of bacterial exotoxin and endotoxin antagonists

Yeast-display;Superantigen;Staphylococcal Enterotoxin C;MD-2;Toll-like receptor 4 (TLR4);Lipopolysaccharide (LPS)

Mattis, Daiva Maria

关键词: Yeast-display;    Superantigen;    Staphylococcal Enterotoxin C;    MD-2;    Toll-like receptor 4 (TLR4);    Lipopolysaccharide (LPS);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/88242/MATTIS-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Gram negative and positive bacteria have evolved toxins to aid in their ability to colonize host organisms. Some gram-positive bacteria produce exotoxins called superantigens that hyper-stimulate the immune system by crosslinking the variable region of the beta chain (Vβ) of T cell receptors with the antigen presenting major histocompatibility complex II molecule on the surface of antigen presenting cells. This hyper-stimulation leads to overproduction of cytokines, which can result in toxic shock. In addition, the action of the superantigens has been implicated in many diseases including necrotizing pneumonia and endocarditis. Gram-negative bacteria produce lipopolysaccharide (LPS), also called endotoxin, as a major constituent in their outer cell walls. LPS binds to the host protein called MD-2 and the LPS:MD-2 complex associates with cell surface homodimeric Toll-like receptor 4 (TLR4). This tri-molecular interaction can lead to massive stimulation of cytokines from TLR4+ antigen presenting cells, resulting in endotoxic-mediated septic shock. This process has also been suggested to play a role in asthma. A lack of therapeutics for both exotoxin and endotoxin induced shock and implicated diseases, as well as an interest in further understanding these molecular interactions, guided my studies and development of high affinity agents to neutralize these toxins.In chapter two, directed evolution was used to engineer a high affinity antagonist against the superantigen Staphylococcal enterotoxin C3 (SEC3). I used a previously error-prone engineered Vβ against SEC3 as a starting template for further engineering. Yeast display was used to create two libraries in two different regions of the previously engineered Vβ to improve its affinity for SEC3. The mutations from the highest affinity mutant selected from each library were combined to create a single mutant that had improved binding to SEC3 over either mutant. The highest affinity Vβ antagonist was tested and found to be effective in various rabbit models with SEC3 by the Schlievert laboratory.Chapter three describes the cross reactivity of the high affinity SEC3 antagonist described in chapter two, with allelic variants of SEC3 (SEC1, SEC2, and SEC4), as well as the highly homologous superantigen, Staphylococcal enterotoxin B (SEB). Residues potentially responsible for the cross reactivity with SEB were mutated and tested for binding to SEB and SEC3. The SEC4 secreting bacteria strain MW2 was used in necrotizing pneumonia and infective endocarditis rabbit models by the Schlievert laboratory that confirmed the in vivo ability of the antagonist to effectively neutralize more than one strain of SEC.In chapter four, MD-2 was expressed on the surface of yeast and shown to bind MD-2 specific monoclonal antibodies and to its ligands, LPS and TLR4. To test the platform, alanine mutants were engineered at residues identified from previous studies that tested for binding to LPS as well as TLR4. The alanine mutants behaved as anticipated based on the previously published literature. Based on the alanine mutant results, six yeast display libraries were engineered in MD-2 in these regions, and a seventh library was made using error-prone PCR. Ligand studies of the MD-2 mutants allowed identification and insight into the role of critical residues in MD-2 stability and ligand binding.