Association of Acinetobacter baumannii EF-Tu with cell surface, outer membrane vesicles, and fibronectin.

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.

Adhesion of acinetobacter baumannii to extracellular proteins detected by a live cell-protein binding assay.

INTRODUCTION: Acinetobacter baumannii is involved in various infectious diseases ranging from nosocomial community-acquired infections to those acquired following war or natural disasters. The treatment has become exceedingly difficult partly because the bacterium can form biofilms. Therefore, it is imperative to elucidate mechanisms of the biofilm formation that may be exploited to develop therapeutic strategies. OBJECTIVE: To develop an assay by which the role of the bacterial extracellular proteins can be studied in mediating cell adhesion and biofilm formation. METHODS: Biofilm mutants of A. baumannii were generated. Proteins from the cell-free spent cultures and outer membrane fractions of the mutants and the wild type strain were characterized by SDS-PAGE based proteomic analysis. The PAGE-based membrane binding assays were developed to examine bacterial adhesion to the released proteins immobilized on the blotting membranes. RESULTS: The mutants exhibited deficiencies in formation of biofilms and in production of the biofilm-related proteins, such as OmpA. A novel PAGE-based membrane binding assay was established, and the results show attachment of the wild type cells to the released proteins in contrast to that of the mutant cells deficient in the outer membrane proteins. The results imply that these mutants have lost the cell surface-associated proteins that mediate cell adhesion to the released proteins. CONCLUSION: This novel assay can be used to study the live bacterial adhesion to extracellular proteins. The results suggest that the outer membrane proteins may mediate cell attachment through binding to the released proteins for biofilm formation.