Molecular basis for the folding and autocatalytic processing of beta-helical autotransporter passenger domains

Abstract

Autotransporters are a distinct class of outer membrane proteins (OMPs) and the largest family of virulence factors produced by Gram-negative bacterial pathogens. The virulence functions of autotransporters depend on their correct assembly at the bacterial cell surface. Assembly requires the C-terminal beta-barrel domain to be inserted into the outer membrane in a partially folded state to facilitate translocation of the N-terminal passenger domain onto the cell surface, where it folds into its biologically active form. Some passenger domains remain attached to their cognate beta-barrel domains to typically function as adhesins or as mediators of aggregation and biofilm formation, while others are cleaved and released into the extracellular environment to function as toxins or in disruption of host immune response. Once translocated, autotransporter passenger domains predominantly fold into elongated beta-helical stalk-like structures that project outwards from the cell surface. Many studies have provided valuable insights into how autotransporters adopt their functional conformations, yet many mechanistic details are lacking.

In this study, we used mutagenesis analysis to test the role of the extracellular loops of the beta-barrel domain belonging to the autotransporter, plasmid-encoded toxin (Pet) in folding of the cognate passenger domain. We found that the fifth extracellular loop (L5), which forms a beta-hairpin structure, is required for Pet passenger folding into its native, protease-resistant conformation in vivo and in vitro. This work revealed that it is the beta-strand propensity of the L5 beta-hairpin, rather than specific amino acid residues, that is important for facilitating passenger folding. Based on these findings, we proposed that L5 mediates Pet passenger folding via beta-augmentation and showed that this folding mechanism is conserved in the Serine Protease Autotransporters of Enterobacteriaceae (SPATE) subfamily. To test if loop-mediated passenger folding is conserved in autotransporters beyond the SPATE subfamily, we used antigen 43 (Ag43) as a model protein. Ag43 is an autotransporter that mediates cell aggregation and biofilm formation. We found that both the fourth and fifth extracellular loops of the beta-barrel domain are required for rapid folding of the passenger domain and for Ag43-mediated bacterial cell aggregation. Together, these findings suggest that loop-mediated passenger folding could be a conserved folding mechanism among autotransporters with a beta-helical structure. We also show that proteolytic processing of Ag43 occurs through autocatalysis, in the absence of host or exogenous proteases, and that autocatalytic processing of the passenger domain from its cognate beta-barrel is not required for Ag43 function.

This study provides new mechanistic understanding of how autotransporter passenger domains fold and undergo proteolytic processing. Our results suggest that the autotransporter beta-barrel domain is a folding vector that mediates passenger domain folding directly via the extracellular loop(s). We show that inhibition of passenger domain folding through deletion of the extracellular loop(s) inhibits autotransporter-mediated virulence functions, and thereby propose that the extracellular loops are potential therapeutic targets in treating and preventing bacterial infections. Show more