Page Analyses of Staphylococcus aureus Host Interactions R01 AI020624 A.
SPECIFIC AIMS Staphylococcus aureus (S. aureus) is a potent, opportunistic human pathogen that has evolved in a symbiotic relationship with its hosts and is notorious for its ability to cause a variety of infections, ranging from relatively mild skin infections to life threatening diseases such as sepsis, pneumonia and endocarditis. Due to the rapid emergence of antibiotic resistant strains (i.e. methicillin resistant S. aureus (MRSA)); novel preventive and therapeutic strategies are being explored. At least nine immune therapeutic or preventive Phase ll/lll clinical trials have failed (1, 2). An important reason for these failures may be the bacteria's ability to evade even an enhanced host's defense system. S. aureus is unique among bacterial pathogens in that it expresses over a dozen cell-wall anchored and secreted fibrinogen (Fg) binding proteins (3) that will recruit and possibly exploit Fg in host evasive strategies. We thus hypothesize A) that Fg recruited by specific cell-wall anchored and/or secreted proteins forms a protective shield surrounding the organism that protects S. aureus from phagocytosis and clearance, and B) that the formation of the shield is critical for the virulence potential of the organism. The goals for the proposed studies are to characterize the different Fg binding interactions involved in the formation of the protective Fg shield in molecular details. A detailed understanding of these interactions will provide a molecular base for the future rational design of anti-staphylococcal immuno-preventive or -therapeutic products. It appears that for these products to be effective they must inhibit the formation of the Fg shield or disrupt an already assembled shield. The following specific aims are proposed: A1: Characterize the complex Fg interactions with S. aureus MSCRAMMs. These studies are ongoing. We earlier identified a group of Fg binding S. aureus MSCRAMMs and demonstrated that many of these proteins use a unique and dynamic binding mechanism to interact with Fg that we called Dock, Lock and Latch (4). We have now shown in preliminary studies that this mechanism represents an initial interaction that appears to be followed by a secondary interaction and that both steps are required for high affinity Fg binding. We propose to characterize these complex interactions in detail. The sub-aims are: i) Determine crystal structures of Clumping factor A (ClfA) in complex with intact Fg and Fg fragments and biochemically verify these structures. ii) Identify natural ClfA variants with altered affinity for Fg. iii) Explore common sequences in ClfA-like staphylococcal MSCRAMMs. iv) Characterize the interaction of Fg with Clumping factor B (ClfB). A2: Characterize Fg binding to small secreted S. aureus proteins S. aureus produces a number of small secreted proteins that include Coagulase (Coa), Extracellular fibrinogen binding protein (Efb), von Willebrand factor binding protein (vWbp), Extracellular matrix binding protein (Emp) and Extracellular adherence protein (Eap). These proteins all bind Fg and can participate in the formation of an Fg containing macrostructure surrounding bacteria in the center of an abscess. The Fg binding sites in the secreted proteins appear to be primarily located to intrinsically disordered section of the proteins and therefore are found in linear sequence segments of the proteins. However, recent studies suggest that also an ordered domain in vWbp binds to a linear sequence in Fg. Preliminary studies of Efb and Coa have outlined a road map for how to identify Fg binding motifs in disordered protein segments and characterize their interactions with Fg. We will now i) Identify the Fg binding sites in vWbp, Emp and Eap, ii) Determine sequences in Fg targeted by ordered domains in small secreted staphylococcal proteins, and iii) Determine the crystal structures of the Efb/Coa Fg binding motifs in complex with Fg fragments. This will allow us to determine the sites in Fg targeted by these proteins. A3: Determine the structure/function relationships in Fg dependent host evasions. We will use a multi approach strategy to seek answers to the following questions: i) Can binding of Fg to a staphylococcal protein induce conformational changes in the Fg molecule? ii) Is there a correlation between a MSCRAMM's Fg affinity and virulence potential? iii) Can we generate specific monoclonal antibodies that cross react with and inhibit Fg binding to several staphylococcal proteins?
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