title: The Haemophilus influenzae Hia autotransporter contains an unusually short trimeric translocator domain.
abstract: Gram-negative bacterial autotransporter proteins are a growing group of virulence factors that are characterized by their ability to cross the outer membrane without the help of accessory proteins. A conserved C-terminal beta-domain is critical for targeting of autotransporters to the outer membrane and for translocation of the N-terminal "passenger" domain to the bacterial surface. We have demonstrated previously that the Haemophilus influenzae Hia adhesin belongs to the autotransporter family, with translocator activity residing in the C-terminal 319 residues. To gain further insight into the mechanism of autotransporter protein translocation, we performed a structure-function analysis on Hia. In initial experiments, we generated a series of in-frame deletions and a set of chimeric proteins containing varying regions of the Hia C terminus fused to a heterologous passenger domain and discovered that the final 76 residues of Hia are both necessary and sufficient for translocation. Analysis by flow cytometry revealed that the region N-terminal to this shortened translocator domain is surface localized, further suggesting that this region is not involved in beta-barrel formation or in translocation of the passenger domain. Western analysis demonstrated that the translocation-competent regions of the C terminus migrated at masses consistent with trimers, suggesting that the Hia C terminus oligomerizes. Furthermore, fusion proteins containing a heterologous passenger domain demonstrated that similarly small C-terminal regions of Yersinia sp. YadA and Neisseria meningitidis NhhA are translocation-competent. These data provide experimental support for a unique subclass of autotransporters characterized by a short trimeric translocator domain.
authors.affiliation: Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. stgeme@borcim.wustl.edu
authors.forename: Neeraj K
authors.initials: NK
authors.lastname: Surana
authors.forename: David
authors.initials: D
authors.lastname: Cutter
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Barenkamp
authors.forename: Joseph W
authors.initials: JW
authors.lastname: St  Geme
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001692:Biological Transport; D015153:Blotting, Western; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D006193:Haemophilus influenzae / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D015007:Yersinia / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; C429644:Hia adhesin; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C064832:YadA protein, Yersinia; D006639:Histidine; D004247:DNA