Steps Towards Investigating the Role of the Putative-Autochaperone Domain in Ag43 Passenger Folding In Vitro

Abstract

Type V secretion systems use autotransporter (AT) proteins to transport material out of the cell without consuming ATP in gram-negative bacteria. These ATs are canonically composed of three units: a signal peptide allowing for movement to the cell-surface, an N-terminal passenger domain housing the effector protein, and a C-terminal translocator domain. Current literature on such systems has identified a conserved region termed the autochaperone regions (AC) at the C-terminus of the passenger domain in many ATs. Furthermore, several ACs have been identified in BrkA, PrtS, EspP, IcsA and AIDA-I, and have been found to serve a role in passenger folding. Ag43 functions to promote intercellular aggregation of Escherichia coli, and possibly biofilm formation. However, the role of AC (amino acids 578 to 692) in Ag43 folding has not been experimentally determined. Our goal was to test whether the AC domain (amino acids 578 to 692) is required for folding of the Ag43 passenger domain. We aimed to generate two plasmids, one containing full-length Ag43 passenger (amino acids 52 to 692) and the other containing a truncated passenger without the AC domain (amino acids 52 to 552). We present an experimental design for the characterization of Ag43 folding. Our study provides a foundation towards understanding the role of the AC (amino acids 578 to 692) in Ag43 folding and highlights some novel and specific challenges of studying this protein.