Abstract
Elongation factor thermo-unstable (EF-Tu), an abundant multifunctional protein, is pivotal during protein synthesis and is an important antigen. Few studies have addressed the role of this protein in Brucella species, and the epitopes of this protein have not been reported. Here, we describe a monoclonal antibody (McAb), BD6, for EF-Tu in Brucella melitensis. Using western blotting involving a series of partially overlapping recombinant EF-Tu truncation peptides, a novel linear B-cell epitope, 110QTREHIL116 (EF), was identified. Alanine-scanning mutagenesis revealed that residues Q110, T111, R112, I115, and L116 were core residues involved in recognition. Sequence alignment suggested that the epitope peptide was conserved among bacterial species but differed by one amino acid residue (I115) from the host sequence. The epitope peptide was recognized by sera from B. melitensis-infected mice, and while recombinant epitope peptide induced a strong humoral immune response, the corresponding mouse peptide, QTREHLL, did not. These results suggested that I115 may be the key residue for the host immune system to distinguish between bacterial and self epitope EF sequences. Indirect immunofluorescence and western blotting assays showed that epitope peptide could be used in Saccharomyces cerevisiae, human embryonic kidney cell (HEK-293), and chicken fibroblast cell (DF1) expression systems and immunoprecipitation assay. Together, our results suggested that the McAb BD6 is a useful tool for further investigation of the potential functions of the EF-Tu protein in pathogen-host interactions, and that the epitope tag may be useful for application as a novel affinity tag to identify other bacterial pathogens, especially convenient for the identification of intracellular bacteria.
Highlights
The protein elongation factor thermo-unstable (EF-Tu) is abundant both in prokaryotic and eukaryotic cells, reaching concentrations almost ten times higher than that of ribosomes under rapid growth conditions (Furano, 1975)
Indirect immunofluorescence assays showed strong immunofluorescence on the surface of yeast cells expressing B. melitensis Elongation factor thermo-unstable (EF-Tu) (Figure 1Ba) but not on those transformed with the empty vector (Figure 1Bb)
EF-Tu proteins from some pathogenic bacteria play a pivotal role in protein synthesis as well as being novel moonlighting proteins involved in many cellular and disease processes (Marques et al, 1998; Alonso et al, 2002; Sharma et al, 2011; Jiang et al, 2016)
Summary
The protein elongation factor thermo-unstable (EF-Tu) is abundant both in prokaryotic and eukaryotic cells, reaching concentrations almost ten times higher than that of ribosomes under rapid growth conditions (Furano, 1975). Recent studies have suggested that the EF-Tu proteins of several prokaryotic plant and animal pathogens are exposed on the cell surface (Balasubramanian et al, 2008; Barel et al, 2008; Wolff et al, 2013; Amimanan et al, 2017) and that surface-located EFTu proteins play a pivotal role in bacterial adhesion, invasion, and host immune evasion (Jonak, 2007; Kunert et al, 2007; Mohan et al, 2014; Amimanan et al, 2017). Brucella melitensis is an important zoonotic pathogen. Wang et al (2013) found that EF-Tu-encoding gene tuf plays an important role in the virulence attenuation of B. melitensis vaccine strain M590. The specific virulence mechanism and other functions of the EF-Tu protein remain unclear, and molecular tools needed to carry out an analysis, including a monoclonal antibody (McAb) targeting the Brucella EF-Tu protein, have yet to be developed
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