Engineering an OmpT Cleavage Site in the BrkA Passenger Domain to Explore the Role of the Conserved Autochaperone Region

Abstract

The secretion of autotransporters stands as a distinctive mechanism employed by Gram-negative bacteria for the transportation of proteins to their cell surface. BrkA is an autotransporter protein in Bordetella pertussis which plays a pivotal role in conferring serum resistance and enhancing the bacterium's adherence to host cells. BrkA comprises three key domains: an N-terminal signal peptide domain, a 73 kDa passenger α-domain, and a 30 kDa translocator β-domain. BrkA secretion has been well studied, and it has been previously shown that the autochaperone region glutamate⁶⁰¹–alanine⁶⁹² is essential for proper protein folding and protection against proteolysis. This region, however, is not necessary for translocation of the protein across the outer membrane. Previous literature has also shown that following translocation out of the cell, cleavage occurs at the asparagine⁷³¹– alanine⁷³² site to dissociate the passenger from the translocator β-domain. However, despite cleavage at the cell surface, the BrkA passenger cannot be detected in the supernatant. It is thought that a non-covalent interaction may be responsible for this phenomenon by anchoring the passenger to the cell membrane but this process remains understudied. In this study, we sought to identify the specific region that may non-covalently interact with the β-barrel region by engineering a secondary OmpT cleavage site before glutamate⁶⁰¹. Two rounds of site-directed mutagenesis were employed to introduce a polyhistidine tag at the N-terminus of the BrkA passenger and then an OmpT cleavage site at glutamate 601 of the BrkA passenger. Western blot analysis of whole cell lysates of an OmpT-expressing strain of E. coli (UT2300) detected a band corresponding with the BrkA passenger (61 kDa) processed at the OmpT cleavage site, suggesting that it remains cell-bound after cleavage.  A 61 kDa band was not observed in Western blots of whole cell lysates in an OmpT deficient strain of E. coli (UT5600).  We did not detect a band of 61 kDa using Western blots of filtered culture supernatants in either strain.  It is also possible that the concentration of the 61 kDa protein moiety was too low in supernatants for Western blot detection or that the cleaved BrkA passenger was unstable in the absence of the autochaperone region and degraded by extracellular proteases in the media.