B-285. Characterization and Expression of a Novel Acylated Bordetella Autotransporter

P. V. Sims, R. C. Fernandez;
Univ. of British Columbia, Vancouver, BC, CANADA.

Autotransporters are Gram-negative bacterial proteins consisting of a signal peptide, passenger domain and C-terminal translocation unit (TU). The TU forms a β-barrel in the outer membrane (OM), while the functional passenger is exported to the surface. Some autotransporters are amino-acylated, such as NalP of Neisseria, AlpA of Helicobacter and SphB1 of Bordetella; however, their secretion is poorly understood. Lipoproteins in Gram-negative bacteria are localized to the OM, via the LolABCDE pathway. We recently identified a putative acylated autotransporter, bapF, from Bordetella genome sequences. The bapF gene was cloned and expressed in E. coli, allowing antibodies to be raised to the BapF passenger. Western blot analysis indicated that bapF is expressed in B. bronchiseptica RB50 only under bvg- conditions (bvg- denotes the “off” state of the major virulence regulon in Bordetella species). We hypothesize that BapF is a functional acylated autotransporter, and have tested this in an E. coli expression system. Processing of BapF into passenger and TU domains was observed, and later confirmed by mass spectrometry; processing is known to occur between amino acids 413-454. Expression of BapF in E. coli also promotes microcolony formation in liquid culture, suggesting outer membrane localization of BapF. Sucrose density centrifugation confirmed the BapF passenger is localized to the OM, and is supported by data showing that BapF partitions to sarcosyl-insoluble fractions along with other membrane proteins. To test whether association to the OM is mediated by acylation we used globomycin to inhibit LspA (a critical enzyme required for acylation). Globomycin at 5μg/ml inhibited BapF biogenesis and dispersed microcolonies, yet was non-lethal to cells. In summary, BapF is expressed in B. bronchiseptica under bvg- conditions. In E. coli, BapF is an OM acylated autotransporter which is processed into passenger and TU domains. This data suggests an interaction between BapF secretion and the LOL pathway, making BapF a potential model protein for investigating secretion of acylated autotransporters.