B-288. Secretion Importance of Conserved Residues in the C Terminus of a Serine Protease Autotransporter Protein

Y. T. Yen1,2, C. Tsang1, T. A. Cameron1, C. Stathopoulos1;
1California State Polytechnic Univ., Pomona, CA, 2Univ. of Houston, Houston, TX.

The serine protease autotransporters of the Enterobacteriaceae (SPATE) constitute a family of conserved, virulence proteins secreted by a number of enteric Gram-negative bacterial pathogens. An autotransporter polypeptide is comprised of three domains: an N-terminal domain required for its inner membrane translocation; an internal passenger domain secreted to the extracellular environment and conferring biochemical function to the bacterium; and a C-terminal translocator domain capable of forming a β-barrel in the outer membrane and mediating the secretion of the passenger domain to the cell surface. Using the prototype SPATE protein Tsh as a model for the study of the autotransporter secretion mechanism, we set out to identify amino acid residues in the β-barrel that might play a role in the secretion of the Tsh passenger. To do so, the sequences of 20 known SPATE autotransporters were aligned and four conserved motifs in the translocator domains were identified. A motif in the α-linker region at the N terminus of the translocator was previously shown by our lab to influence Tsh passenger secretion. In this study, we focused on evaluating the secretion role of the remaining three motifs, which include 24 highly conserved residues located in the barrel-forming region of the Tsh translocator domain. Each of these positions was mutated by PCR-based site-directed mutagenesis. The mutant constructs were then subjected to cell fractionation, and the protein secretion levels in the culture medium, outer membrane, periplasm, and on the cell surface were assessed by various immunochemical techniques. When compared to wild-type Tsh, more than half of the mutants exhibited secretion defective phenotypes, including diminished secretion into the culture medium, cell lysis, and protein accumulation in different compartments of the cell envelope. The data suggest the function of these residues in various steps of Tsh’s passenger secretion process, including barrel formation and insertion, outer-membrane translocation, and processing. The results hence contribute to a better understanding of the role of C-terminal β-barrel in the autotransporter protein secretion mechanism.