Pathogenic bacteria have the ability of killing the host cells they infect. Knowledge of apoptosis-inducing virulent factors and information about their mechanism of action could provide the basis for the design of strategies to attenuate bacterial diseases. The protein cascade used by human macrophages to detect the presence of pathogens is the Toll-like receptor pathway that via its key cytoplasmic mediator the TIR domain containing protein MyD88 ends with the activation of the transcription factor NF-kappaB. Analyzing DNA data coming from sequencing the full length genome of pathogenic bacteria, like Staphylococcus aureus (Sa), Salmonella enteriditis (Se) and Eschericia coli (Ec), several TIR domain containing proteins have been annotated. Our collaborator results indicate that prokaryotic and eukaryotic TIR domain containing proteins interact generating an unproductive complex for activation of the innate immune response of the host. The objective of the proposed studies is to investigate the molecular mechanisms underlying the regulatory and targeting interactions between prokaryotes and eukaryotes TIR domains, using structural and biophysical techniques. Threading methods permit the folding of these bacterial sequences into bona fide TIR domains. We will apply X-ray crystallography and NMR to determine the atomic structure of the Ec-TIR domain. We plan to analyze the chemical nature of the interaction with its binding partner, the eukaryotic MyD88-TIR domain, utilizing surface plasmon resonance and isothermal calorimetry. Once suitable conditions for complex formation are identified, the three-dimensional structure of Ec-TIR bound to MyD88-TIR will be solved. The applicant expects that these studies will reveal the first structural and functional information about TIR signaling by bacteria in humans. Ultimately, it may provide insights for the development of pharmaceutical agents that can interfere specifically with bacterial TIR signaling events whose actions in infected human cells lead to apoptotic processes.
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