An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure

Abstract

Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, 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. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA’s passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA’s higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA’s nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces.

On‐fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin

“…The His‐tag was inserted into the predicted loop region originally located at the tip of the AtaA fiber before the N‐terminal Ylhead domain (Figure ). The fiber lengths could be estimated from purified AtaA fiber fragments (3CAtaAs; Yoshimoto et al, ), as shown in Figure (between the N‐terminus and FGG_5: 225 nm, between the N‐terminus and FGG_2: 180 nm, and between FGG_2 and FGG_5: 45 nm). Thus, HisA, ΔNHisB, and ΔNHisC were designed to generate the fibers with 225, 135, and 45 nm in lengths, respectively, on the host cells.…”

“…The distances between the cell surface and the colloidal gold on the fibers (20 fibers), which were selected from ones localizing at the farthest places from the edge of cell surface on 2D TEM images, were measured as the actual fiber length. The general procedures of TEM followed the methods previously described (Yoshimoto et al, ).…”

“…Although cell surface display systems using monomeric autotransporters have been developed, called “autodisplay,” TAAs have never previously been used for surface display, to the best of our knowledge, except for AtaA, the TAA from Acinetobacter sp. Tol 5 (Ishikawa, Nakatani, et al, ), in which a proteolytic cleavage site was inserted and displayed for the isolation of the TAA fibers (Yoshimoto, Nakatani, Iwasaki, & Hori, ).…”

“…AtaA, one of the largest TAAs, consists of polypeptides with 3,630 amino acids and forms a long, peritrichate nanofiber 225 nm in length for its PSD, and its fiber structure is stable in a wide range of thermal and pH conditions (Yoshimoto et al, ). Thus, AtaA could possibly have advantages over conventional surface display systems because it can stably display functional molecules at an appropriate site distant from the cell surface.…”

Cell behavior of the highly sticky bacterium Acinetobacter sp. Tol 5 during adhesion in laminar flows

“…Tol 5, was previously isolated from a biofiltration process 14 . This bacterium shows high adhesiveness to various abiotic surfaces from hydrophobic plastics to hydrophilic glass and stainless steel, and also demonstrates autoagglutination through its peritrichate fiber protein AtaA 15 – 17 . AtaA is a member of the TAA family, which contains proteins that are usually involved in bacterial adhesion to host cells and extracellular matrix proteins such as collagen and fibronectin, as well as in autoagglutination, colonization, biofilm formation, and serum resistance 18 – 23 .…”