UT5600 alone (?) was used as a negative control

UT5600 alone (?) was used as a negative control. trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity. Microbial adherence to host tissues is an essential early step in the pathogeneses of infectious diseases (18). High-affinity adherence is important in allowing microorganisms to overcome normal host protective mechanisms associated with mechanical force, such as peristalsis, mucociliary clearance, and coughing. Typically, the process of adherence involves a Montelukast specific interaction between a microbial surface protein, called an adhesin, and a complementary Montelukast host cell receptor. Autotransporter proteins are a large family of extracellular proteins that are present in a number of gram-negative pathogenic bacteria ER81 (16). These proteins are synthesized as precursor proteins with three functional domains, an N-terminal signal peptide, an internal passenger domain, and a C-terminal pore-forming translocator domain (17, 19, 27). The C-terminal translocator domain is embedded in the outer membrane and facilitates delivery of the internal passenger domain to the bacterial surface. The trimeric autotransporters represent a Montelukast subfamily of autotransporter proteins and are defined by the presence of a very short C-terminal translocator domain that forms highly stable trimers in the outer membrane (30, 39). Thus far, all characterized members of the trimeric-autotransporter subfamily have been found to possess adhesive activity, in most cases mediating bacterial adherence to eukaryotic cells (4, 6-8, 31, 35) or extracellular matrix proteins (25, 41) and in some cases resulting in binding of circulating factors, such as immunoglobulins or complement components (1, 13, 33, 34, 42). The prototype members of the trimeric autotransporter subfamily are the Hia adhesin and the YadA protein. Hia mediates high-affinity adherence to respiratory epithelial cells (23), and YadA is a virulence factor capable of mediating adherence to host cells and extracellular matrix proteins and involved in serum resistance (14). In experiments with Hia, the Montelukast C-terminal 76 amino acids were capable of presenting a functional heterologous passenger domain on the bacterial surface (39). Similarly, in studies of YadA, the C-terminal 70 amino acids were sufficient for translocating an N-terminal FLAG epitope across the outer membrane (30). Further biochemical analysis established that the C termini of both Hia and YadA form heat-resistant, sodium dodecyl sulfate-resistant trimers in the outer membrane and that Hia requires formic acid denaturation for dissociation (30, 39). Consideration of this information in combination with secondary-structure predictions has led to the proposal that the C-terminal translocator domains of both Hia and YadA form 12-stranded pore-forming -barrels containing four strands from each of three subunits (30, 39). The passenger domains of all three subunits are believed to be translocated to the bacterial cell surface through the trimeric pore (9). Recent crystal structures of the Hia primary binding domain (HiaBD1) and the YadA collagen-binding domain have established that the passenger domains of these proteins are capable of trimerization independent of the translocator domain (28, 43). HiaBD1 is an intricately folded trimer with a large hydrophobic core and multiple subunit-subunit interactions. The adhesive activity of this domain maps to an acidic pocket that is formed by a single monomer and is present on all three faces of the trimer (three pockets per trimer). The YadA collagen-binding domain is a novel left-handed parallel -roll with a trimeric architecture and potential to interact with three separate collagen fibers. In the present study, we examined the relationship between trimerization and the capacity for adhesive activity in the Hia and YadA passenger domains. We found that subunit-subunit interactions and stable trimer formation are essential for native folding and stability and ultimately for full-level adhesive activity. This study provides important insights into the structure and function of trimeric autotransporters and suggests that disruption of the trimeric architecture of these proteins may be an effective strategy to eliminate biological activity. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. strains DH5 (Life Technologies), BL21(DE3), and XL-1 Blue have been described previously (32). strain UT5600 is an OmpT? OmpP? derivative of strain RW193 (12). Plasmid vectors used in this study include pACYC184 (New England BioLabs), pT7-7 (40), and pHAT10 (Clontech). The plasmid pMW10 (also referred to as pYadA) contains the gene from strain O:8 and was a gift from Virginia Miller (Washington University, St. Louis, MO). The plasmid pHMW8-7 (also referred to as pHia) contains the gene from strain 11 in pT7-7 (4). The plasmid pHMW8-7BS2 (also referred to as pHiaBS2).