Long-term goals are to understand the pathogenesis of methicillin-resistant Staphylococcus aureus (MRSA) that secrete superantigens (SAgs) such as enterotoxin (SE) B or C, SE-like Q, and toxic shock syndrome toxin-1 (TSST-1) and Bacillus anthracis. MRSA are emerging pathogens and their SAgs category B select agents. B. anthracis is a category A select agent. We have assembled two projects that are described below. Project 1. High-Affinity Molecules to Inhibit Superantigen Function. When produced in vivo or when purposely administered, SAgs are exceptionally toxic (TSS symptoms at doses of 0.1 ng/human). Most MRSA associated with post-influenza TSS, necrotizing pneumonia, and purpura fulminans produce SEB, SEC, SE-like Q, and TSST-1.
Specific Aim 1. To engineer and characterize V(3-T cell receptor (TCR) regions that bind with high affinity to SAgs and neutralize them, most importantly SEB, SEC, SE-like Q, and TSST-1.
Specific Aim 2. To engineer and characterize MHC II regions that bind with high affinity to SAgs and neutralize their activities.
Specific Aim 3. To test the ability of soluble Vp-TCRs and MHC Ms to neutralize the activities of SAgs in vitro and in vivo in rabbits. Project. 2: Inhibitors of SrrA-SrrB and BrrA-BrrB Regulated Exotoxin Production. MRSA require oxygen for exotoxin production. This observation led to identification of a """"""""Master Switch"""""""" two-component system required for oxygen control of exotoxin production in MRSA. This system, designated srrA-srrB, represses exotoxin production and virulence under conditions of low oxygen. Recently, a homolog in B. anthracis was described-BrrA-BrrB. BrrA-BrrB functions to induce anthrax toxin genes and facilitate disease production.
The aims will provide a foundation for interfering with diseases by developing small molecule inhibitors.
Specific Aim 4. To characterize SrrB and BrrB as histidine kinases with ability to phosphorylate SrrA and BrrA.
Specific Aim 5. To evaluate small molecule inhibitors of SrrB/BrrB to prevent production of SAgs, anthrax toxin, and B. anthracis capsule.
Aim 6. To determine the 3-D structures of SrrB and BrrB proteins or extracellular/intracellular domains and combined with menaquinone or inhibitors.
Highly antibiotic resistant Staphylococcus aureus are the most significant invasive pathogens in the United States. Bacillus anthracis and staphylococcal exotoxins (anthrax toxins and superantigens) are highly toxic to humans. We propose to engineer molecules to neutralize Staphylococcus aureus exotoxins and identify small molecule inhibitors that prevent production of staphylococcal and Bacillus anthracis exotoxins.
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