Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Autotransported virulence factors are frequently employed by Gram-negative bacteria to augment one or more stages of pathogenesis. Autotransporters contain an N-terminal passenger domain that encodes the virulence effector functions, and a C-terminal β-barrel that is required in passenger translocation across the outer membrane and to the bacterial cell surface. Passengers are extremely diverse in their sequence, size, and effector activity. There also appears to be a high level of mixing of conserved subdomains, motifs, and repeats in the construction of these passengers. For these reasons it is still poorly understood how these diverse passengers are efficiently translocated to the surface. Recently, we have characterised a conserved 32-aa motif called the Passenger-associated Transport Repeat (PATR; PF12951) that is found in thousands of passenger sequences. The PATR contains four highly conserved glycine residues, and upon individual substitution of these residues using the model autotransporter IcsA from Shigella flexneri, we observed reduced levels of the passenger on the bacterial cell surface using quantitative florescence microscopy. Reduced levels of passenger activity for PATR-mutants were observed in tissue culture infection models. This occurred despite the equivalent localisation of PATR-mutants to outer membrane fractions comparatively to wild-type IcsA. In vivo limited-proteolysis and pulse-chase proteolysis assays revealed striking reductions in the rate of mature PATR-mutant surface deposition. Using database mining methods we show that PATR-containing autotransporters: (i) have distinct virulence functional features, (ii) have significantly larger passengers than non-PATR autotransporters, and (iii) have distinctly different architectures compared to non-PATR passengers. Further, PATR-type autotransporters have low pIs that cluster by length indicating a possible secretion driving force for large autotransporters. We have also observed differences between Genera in the usage of the PATR-type versus non-PATR autotransporters. Collectively, these results show the importance of the PATR in passenger secretion efficiency, and advance our understanding of autotransporter composition.