TPS domain folding causes activation of a newly defined hemolytic functional domain

Proteus mirabilis is a pathogenic opportunistic Gram-negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a two-partner secretion (TPS) pathway to simultaneously transport and activate HpmA across its outer membrane and release it into the blood of a host where it destroys red blood cells (hemolysis). Like all other TPS proteins, HpmA contains an N-terminal TPS domain (≈300 residues) essential for secretion and a separate C-terminal functional domain (>1000 residues) responsible for virulent functions. To understand the functional relationship between these domains and define the boundaries of the virulent hemolytic domain in HpmA, we made N-terminal, internal, and C-terminal deletions. In the absence of secretion, the unfolded full-length HpmA requires a folded TPS domain to act as a template protein to achieve its hemolytically active form. Using a Template Assisted Hemolytic Activity (TAHA) assay, we can quantitate the effect of the deletions on HpmA function. Unfolded proteins lacking a TPS domain at the N-terminus show no activity in TAHA assays. This suggests that TPS domain folding is required for activation of hemolytic activity. To determine if there is a direct linkage between TPS domain folding and hemolytic activation, we created internal deletions immediately C-terminal to the TPS domain. These demonstrate how TPS domain folding is coupled to activation of hemolytic function. We also show that a significant segment (>300 residues) can be removed from the C-terminus and not affect the hemolytic function. These results have allowed us to define the C-terminal boundary of the hemolytic functional domain. Results of this research have allowed us to locate regions of HpmA necessary for its hemolytic function and characterize how TPS domain folding is required to induce hemolytic activation. These studies have expanded our working model for HpmA secretion, activation, and function. Support or Funding Information This research was generously supported by a University of Wisconsin - La Crosse Undergraduate Research and Creativity Grant (GMW). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.