Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

Abstract

Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

Elongation factor Tu is a multifunctional and processed moonlighting protein

“…Ef-Tu is a multifunctional protein in higher order eukaryotes 11 – 16 , parasites 17 – 20 , fungi 21 and it is has been identified on the surface of a wide range of Gram positive and Gram negative pathogenic and commensal bacteria that associate with metazoan species 2 , 22 – 29 . Bacterial Ef-Tu interacts with nucleolin 30 , 31 , fibrinogen and factor H 23 , 26 , plasminogen and several complement factors 26 , 27 , 32 , laminin 33 , CD21 34 , fibronectin 2 , 33 , 35 , 36 , is immunogenic 37 and adheres to the surface of Hep-2 cells 33 underscoring the multifunctional adhesive characteristics that have been assigned to this molecule. Ef-Tu binds sulfated carbohydrate moieties found on glycolipids and sulfomucin and promotes the binding of Lactobacillus reuteri to mucosal surfaces indicating that Ef-Tu can interact with carbohydrates 38 .…”

“…Ef-Tu binds sulfated carbohydrate moieties found on glycolipids and sulfomucin and promotes the binding of Lactobacillus reuteri to mucosal surfaces indicating that Ef-Tu can interact with carbohydrates 38 . Notably, antibodies against Ef-Tu are induced during infections caused by Staphylococcus aureus 39 , 40 Mycoplasma capricolum 41 , Mycoplasma ovipneumoniae 37 , Chlamydia trachomatis 42 , Burkholderia pseudomallei 43 and Mycoplasma hyopneumoniae 44 . Ef-Tu has been identified in six surfacome studies (excludes cell membrane and envelope isolations) 45 – 50 performed on S. aureus and Ef-Tu is one of twelve proteins consistently identified in the exoproteome of S. aureus from patients with bacteraemia 51 .…”

Quantitative Proteomic Analyses of a Pathogenic Strain and Its Highly Passaged Attenuated Strain ofMycoplasma hyopneumoniae

“…Adherence to ciliated respiratory epithelium is a multifactorial process that also involves other proteins. In our proteomic data, we detected several upregulated moonlighting proteins in strain 168 that could be used to invade host cells: elongation factor Tu [30,31], enolase [29,32,34], and endo-1,4-beta-glucanase [33]. Intensive reports have described some bacterial metabolic enzymes not only performing key metabolic functions in the cytosol of bacterial cell but also locating on the bacterial surface without a signal sequence, moonlighting as an adhesion contributor to host cells [55].…”

“…Within the virulent strain 168 samples, endo-1,4-beta-glucanase, enolase, and translation elongation factor Tu were more abundant, which might conduce to the virulence of M. hyopneumoniae. All these three proteins are cytosolic enzymes, lacking transmembrane domains or traditional signal sequences (Table 3), yet intensive researches during the past years have suggested that they could be detected on microbe surface and moonlight as adhesins [29][30][31][32][33][34]. On one hand we expect to obtain sera to validate the comparative results and on the other hand we are calculated to conduct further experiments around these proteins.…”

Anti‐EF‐Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae

“…Our data support the previous findings that EF‐Tu from Mycoplasma ovipneumoniae and Neisseria gonorrhoeae is immunogenic and elicits a significant immune response in mice. These results are also consistent with previous work that has shown that antisera from mice immunized with recombinant EF‐Tu achieved significant growth inhibition in vitro of the sheep pathogen M. ovipneumoniae .…”