AatA Is a Novel Autotransporter and Virulence Factor of Avian Pathogenic Escherichia coli

Autotransporters (AT) are widespread in Gram-negative bacteria, and many of them are involved in virulence. An open reading frame (APECO1_O1CoBM96) encoding a novel AT was located in the pathogenicity island of avian pathogenic Escherichia coli (APEC) O1''s virulence plasmid, pAPEC-O1-ColBM. This 3.5-kb APEC autotransporter gene (aatA) is predicted to encode a 123.7-kDa protein with a 25-amino-acid signal peptide, an 857-amino-acid passenger domain, and a 284-amino-acid β domain. The three-dimensional structure of AatA was also predicted by the threading method using the I-TASSER online server and then was refined using four-body contact potentials. Molecular analysis of AatA revealed that it is translocated to the cell surface, where it elicits antibody production in infected chickens. Gene prevalence analysis indicated that aatA is strongly associated with E. coli from avian sources but not with E. coli isolated from human hosts. Also, AatA was shown to enhance adhesion of APEC to chicken embryo fibroblast cells and to contribute to APEC virulence.The autotransporter (AT) proteins are a large and diverse family of extracellular virulence proteins of Gram-negative bacteria. All ATs share the same general structure and are comprised of three domains: an amino-terminal signal peptide; an α or passenger domain, which confers the function of the secreted protein; and a C-terminal β domain that mediates secretion through the outer membrane. The cardinal feature of conventional ATs is a long C-terminal translocator domain consisting of about 300 amino acids, in contrast to the very short C-terminal translocator domain (about 70 amino acids) of trimeric ATs that form highly stable trimers in the outer membrane (8). While all trimeric AT proteins identified so far display adhesive activity mediating bacterial interactions with either host cells or extracellular matrix (ECM) proteins, the conventional ATs that have been characterized to date have diverse functions, including adhesion, cytotoxicity, and lipase or protease activity (3, 6, 7, 46, 49, 54).Temperature-sensitive hemagglutinin (Tsh) was the first AT described in avian pathogenic Escherichia coli (APEC), a pathogen which causes extraintestinal infections in turkeys, layers, and broilers (44). This conventional AT, which is encoded by a virulence plasmid, occurs as a 106-kDa extracellular protein and a 33-kDa outer membrane protein. Its passenger domain contains a 7-amino-acid serine protease motif that includes the active-site serine (S²⁵⁹), which has also been found in the secreted domain of IgA1 protease. Although Tsh did not show any IgA protease activity in vitro (51), it was involved in virulence through mediation of APEC''s adherence to the air sacs of chickens (11). The gene encoding a second serine protease AT, termed the vacuolating autotransporter or Vat, was identified in a pathogenicity island (PAI) adjacent to the thrW tRNA gene in APEC (42). Vat has vacuolating cytotoxic activity similar to that of VacA of Helicobacter pylori and contributes to APEC virulence (48). Both tsh and vat are present in E. coli from avian sources and are also found in E. coli isolated from human hosts. In the present study, we identified and characterized a novel AT that is strongly associated with avian E. coli. This AT is encoded by the APEC autotransporter gene (aatA), which has been localized to the PAI found in the virulence plasmid (pAPEC-O1-ColBM; accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_009837","term_id":"157418083","term_text":"NC_009837"}}NC_009837) of APEC O1, the first APEC strain to be completely sequenced (25, 26).