AatA is a novel autotransporter and virulence factor of avian pathogenic Escherichia coli

Abstract

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 beta 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.

FIG. 1.

(A) Diagram showing the conserved domains in AatA. The signal peptide, passenger domain, and translocator domains are indicated; the conserved domains that are similar to protein family PRKD9707 and known AT AidA-I domains are also indicated. (B) Predicted 3D structure of AatA. Coils, strands, and loops are green, yellow, and magenta, respectively,. The translocator and passenger domains are indicated. The hydrophilic core of the translocator domain is approximately 30 Å long and has a diameter of approximately 25 Å; the parallel strands form an 80-Å rod-like shape in the middle of the passenger domain, and the loops form the C-terminal head.

FIG. 2.

Characterization of AatA. (A) SDS-PAGE analysis demonstrating the localization of AatA in APEC O1. Protein samples 2, 3, and 4 were obtained from the membrane protein preparation, and protein samples 5 and 6 were obtained from the outer membrane preparation. Lane 1, protein ladder; lane 2, APEC O1 wild-type strain; lane 3, APEC O1 M_aatA; lane 4, APEC O1 p2 induced by arabinose; lane 5, APEC O1 p1 induced by arabinose; lane 6, APEC O1 p2 induced by arabinose. The arrow indicates the position of AatA. (B) Western blot analysis. Outer membrane protein preparations (lane 1, induced APEC O1 p2; lane 2, induced APEC O1 p1) and membrane preparations (lane 3, induced APEC O1 p2; lane 4, APEC O1 M_aatA; lane 5, wild-type strain APEC O1) were probed with anti-AatA serum.

FIG. 3.

Surface localization of the AatA passenger domain. Immunofluorescence assays with AatA from wild-type strain APEC O1, mutant strain APEC O1 M_aatA, and APEC O1 p2 were performed in the presence of 0.2% arabinose. Bacteria were fixed and incubated with anti-AatA serum, and this was followed by incubation with a secondary polyclonal goat anti-rabbit serum coupled to Alexa 488 (A, D, and H) or DAPI (B, E, and I). (C, F, and J) Merged images. (G) Magnification of the area in the box in panel F. Scale bars = 10 μm.

FIG. 4.

Antibody titers for infected and noninfected chickens. Sera from 10 chickens infected with APEC O1 (group 1) and from 10 chickens inoculated with PBS (control) (group 2) and preabsorbed sera obtained using whole-cell E. coli antigens from the mutant strain (APEC O1 M_aatA) (group 3) were used to detect antibody against AatA with an ELISA. There were significant differences (P < 0.01) among the antibody titers for groups 1 and 2 and for groups 2 and 3, but no significant differences were detected for groups 1 and 3.

FIG. 5.

Adherence assay. The capacities of wild-type strain APEC O1, mutant strain APEC O1 M_aatA, APEC O1 p1 induced by arabinose, and strain APEC O1 p2 induced by arabinose to adhere to CEF cells were compared. The bars indicate the averages of three independent experiments, and the error bars indicate the standard errors. The differences in adherence capacity were significant (P < 0.05).