Determining the Mechanism of Hemolytic Function and Type Vb Secretion by a Gram‐Negative Pathogen

Abstract

Proteus mirabilis is a pathogenic opportunistic Gram‐negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a type Vb, or two‐partner (TPS), secretion system 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 (>1200 residues) responsible for virulent functions. Our goals are to determine how secretion of HpmA is prompted by its folding and identify regions of HpmA critical to its hemolytic function. 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. We term these Template Assisted Hemolytic Activity (TAHA) assays. Performing TAHA assays with genetically altered HpmA, we have quantitated the importance of different regions on HpmA folding and function. We have determined the borders of the hemolytic functional domain, marking the first identification of a functional region on a TPS hemolysin. Other hemolytic proteins are known to adopt a modular method of function. To determine if the function of HpmA is dictated by similar modules, we have created small deletions and amino acid randomizations within the hemolytic functional region. Separately, by rationally designing deletions and variants within the TPS domain of full‐length HpmA, we have determined that the folded template recognizes specific sequences within the unfolded TPS domain to initiate folding. This supports our N‐terminal first secretion model and suggests a continuity of structure and linkage of folding between the TPS domain and the remainder of HpmA. A continuous β‐helical structure starting from the TPS domain could explain this link between domains of secretion and function. To test this link and characterize formation of secondary structure during templated folding, we have performed structural studies using CD spectroscopy. By identifying regions necessary for hemolysis and characterizing how TPS domain folding is required to induce hemolytic activation, our results have expanded our working model for HpmA secretion and function.Support or Funding InformationThis research was supported by an Undergraduate Research and Creativity grant and a Dean's Distinguished Summer Fellowship to GW.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.