The Activator of Apoptosis Smac-DIABLO Acts as a Tetramer in Solution

Smac-DIABLO in its mature form (20.8 kDa) binds to baculoviral IAP repeat (BIR) domains of inhibitor of apoptosis proteins (IAPs) releasing their inhibitory effects on caspases, thus promoting cell death. Despite its apparent molecular mass (∼100 kDa), Smac-DIABLO was held to be a dimer in solution, simultaneously targeting two distinct BIR domains. We report an extensive biophysical characterization of the protein alone and in complex with the X-linked IAP (XIAP)-BIR2-BIR3 domains. Our data show that Smac-DIABLO adopts a tetrameric assembly in solution and that the tetramer is able to bind two BIR2-BIR3 pairs of domains. Our small-angle x-ray scattering-based tetrameric model of Smac-DIABLO/BIR2-BIR3 highlights some conformational freedom of the complex that may be related to optimization of IAPs binding.     

Role of Extracellular Structures of Escherichia coli O157:H7 in Initial Attachment to Biotic and Abiotic Surfaces

Infection by human pathogens through the consumption of fresh, minimally processed produce and solid plant-derived foods is a major concern of the U.S. and global food industries and of public health services. Enterohemorrhagic Escherichia coli O157:H7 is a frequent and potent foodborne pathogen that causes severe disease in humans. Biofilms formed by E. coli O157:H7 facilitate cross-contamination by sheltering pathogens and protecting them from cleaning and sanitation operations. The objective of this research was to determine the role that several surface structures of E. coli O157:H7 play in adherence to biotic and abiotic surfaces. A set of isogenic deletion mutants lacking major surface structures was generated. The mutant strains were inoculated onto fresh spinach and glass surfaces, and their capability to adhere was assessed by adherence assays and fluorescence microscopy methods. Our results showed that filament-deficient mutants bound to the spinach leaves and glass surfaces less strongly than the wild-type strain did. We mimicked the switch to the external environment—during which bacteria leave the host organism and adapt to lower ambient temperatures of cultivation or food processing—by decreasing the temperature from 37°C to 25°C and 4°C. We concluded that flagella and some other cell surface proteins are important factors in the process of initial attachment and in the establishment of biofilms. A better understanding of the specific roles of these structures in early stages of biofilm formation can help to prevent cross-contaminations and foodborne disease outbreaks.