Abstract
Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions.
Highlights
In autotransporters, extracellular proteins in diverse Gramnegative bacteria, the transmembrane anchor domain (TM)2 hosts the autotransport function, called type V secretion, a process in which the passenger domain (PSD) is exported to the bacterial cell surface through a pore formed by the TM [1], with the assistance of periplasmic chaperones and the -barrel assembly machinery [2]
The genus Acinetobacter is ubiquitously distributed in nature, such as in humans, animals, activated sludge, soil, water, 2 The abbreviations used are: TM, transmembrane anchor domain; Trimeric autotransporter adhesins (TAA), trimeric autotransporter adhesin; PSD, passenger domain; CPSD, C-terminal passenger domain; C-terminal Ylhead domain (Chead), C-terminal head; Cstalk, C-terminal stalk; aa, amino acid; inframe deletion (IFD), in-frame deletion; ECM, extracellular matrix; PDB, Protein Data Bank; PS, polystyrene; Ylhead, YadA-like head; Nhead, N-terminal Ylhead domain; Nstalk, N-terminal stalk; ccw, counter-clockwise; CLSM, confocal laser scanning microscopy
Each polypeptide chain of AtaA comprises 3630 amino acid residues, and the homotrimer of the signal peptide-eliminated polypeptide forms a common configuration that can be broadly divided into five regions as follows: an N-terminal Ylhead domain (Nhead; 108 –315 aa); an N-terminal stalk (Nstalk; 316 –2904 aa); a C-terminal Ylhead domain (Chead; 2905–3169 aa); a C-terminal stalk (Cstalk; 3170 –3561 aa); and a TM (3562–3630 aa) (Fig. 1A)
Summary
Construction of Plasmids—Primers used in this study are listed in supplemental Table 1. To construct AtaA recombinant protein fragments for crystallization, encoding regions of DNA were subcloned into the E. coli expression vector pIBAGCN4triHis [34]. To generate an anti-CheadCstalk antibody, a DNA fragment for AtaA(3017–3542) (CheadCstalkX) was subcloned between the BamHI and XhoI sites of pET28d. From pTA2-AtaA_Cter containing a DNA fragment for AtaA(2842–3630), ⌬CheadCstalk, ⌬Chead, ⌬Cstalk, and ⌬Cstalk were generated by inverse PCR using primers (supplemental Table 1) for deleting DNA regions for AtaA(2906 –3475), AtaA(2907–3172), AtaA(3170 –3475), and AtaA(3177–3475), respectively. Cells were lysed in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl for AtaA recombinant proteins or 300 mM NaCl for YadA_head, 20 mM imidazole) at 1000 bar by a French press (SPX, Charlotte, NC) followed by sonication for 20 min (Pw, 7; duty, 30%; Branson Sonifier, Emerson Electric, Louis). Rabbit polyclonal anti-Nhead, anti-CheadCstalk, and anti-Chead antibodies were generated against synthesized polypeptide AtaA[225–241] in Nhead, purified AtaA(3017–
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
