Association ofAcinetobacter baumanniiEF-Tu with Cell Surface, Outer Membrane Vesicles, and Fibronectin

Shatha F Dallo,James Denno,Williams Haskins,Anyu Tsai,Bailin Zhang,Soonbae Hong,Jing Yong Ye,Tao Weitao

doi:10.1100/2012/128705

Abstract

A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu) with bacterial surface. Here we investigated it with Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs) was revealed by immune electron microscopy, and its presence in the outer membrane (OM) and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR) configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin.

Highlights

A Gram-negative and obligate aerobic bacterial species, Acinetobacter baumannii, has emerged as one of the most important nosocomial pathogens [1,2,3,4], raising risks regional and global in the aftermath of war and natural disasters
The identity of recombinant EF-Tu (rEF-Tu) was confirmed with proteomic analysis as we described before [9]
The results revealed by immune transmission electron microcopy (TEM) show that elongation factor Tu (EF-Tu) was physically associated with the outer membrane vesicles (OMVs) and the cell surface

Summary

Introduction

A Gram-negative and obligate aerobic bacterial species, Acinetobacter baumannii, has emerged as one of the most important nosocomial pathogens [1,2,3,4], raising risks regional and global in the aftermath of war and natural disasters. Proteins were extracted from whole-cell lysate, outer membrane (OM) fractions, and cell-free spent cultures (CFCs) of the wild-type and the biofilm mutants of A. baumannii we isolated [9]. Translation elongation factor (EF-Tu) of A. baumannii was detected in cell-free cultures, the data suggesting release of EF-Tu from. Eubacterial EF-Tus belong to the superfamily of GTP-binding proteins It is not a membrane protein, since EF-Tu lacks a signal sequence and transmembrane domains that mediate protein translocation across cell membrane. The bacterial surface association of EF-Tu has been further evidenced by EF-Tu involvement in Staphylococcus aureus biofilm development [16], in mediating attachment to human cells by Lactobacillus johnsonii [17] or P. aeruginosa [18]. We employed a combination of transmission electron microcopy (TEM), proteomics, Western blot, and an optical sensor to show that EF-Tu is associated with OMVs and OM and binds to the host extracellular matrix protein fibronectin

Results

Discussion

Experimental Procedures