Abstract
The metabolic inhibition (MI) test is a classic test for the identification of mycoplasmas, used for measuring the growth-inhibiting antibodies directed against acid-producing mycoplasmas, although their mechanism still remains obscure. To determine the major antigens involved in the immune killing of Mycoplasma bovis, we used a pulldown assay with anti-M. bovis antibodies as bait and identified nine major antigens. Among these antigens, we performed the MI test and determined that the growth of M. bovis could be inhibited effectively in the presence of complement by antibodies against specifically membrane protein P81 or UgpB in the presence of complement. Using a complement killing assay, we demonstrated that M. bovis can be killed directly by complement and that antibody-dependent complement-mediated killing is more effective than that by complement alone. Complement lysis and scanning electron microscopy results revealed M. bovis rupture in the presence of complement. Together, these results suggest that the metabolic inhibition of M. bovis is antibody-dependent complement-mediated killing. This study provides new insights into mycoplasma killing by the complement system and may guide future vaccine development studies for the treatment of mycoplasma infection. Furthermore, our findings also indicate that mycoplasmas may be an appropriate new model for studying the lytic activity of membrane attack complex (MAC).
Highlights
The metabolic inhibition (MI) test is a classic test for the identification of mycoplasmas, used for measuring the growth-inhibiting antibodies directed against acid-producing mycoplasmas, their mechanism still remains obscure
This report describes the production of a rabbit polyclonal antibody that inhibits the growth of M. bovis in vitro and the identification of major antigens recognized by the pAb, determined using a pulldown assay with anti-M. bovis antibodies as bait
At least nine protein bands were visible clearly for lanes that contained M. bovis lysate or lipid-associated membrane proteins (LAMPs) incubated with rabbit anti-M. bovis antibodies compared with the lanes that contained samples incubated with rabbit normal antibodies (Fig. 1)
Summary
The metabolic inhibition (MI) test is a classic test for the identification of mycoplasmas, used for measuring the growth-inhibiting antibodies directed against acid-producing mycoplasmas, their mechanism still remains obscure. To determine the major antigens involved in the immune killing of Mycoplasma bovis, we used a pulldown assay with anti-M. bovis antibodies as bait and identified nine major antigens Among these antigens, we performed the MI test and determined that the growth of M. bovis could be inhibited effectively in the presence of complement by antibodies against membrane protein P81 or UgpB in the presence of complement. Mycoplasmas are a group of bacteria belonging to the class Mollicutes; they lack cell walls, have the smallest bacterial genomes, and presumably evolved from Grampositive bacteria via degenerative evolution [1] Despite their apparent simplicity, over 200 mycoplasma species have been identified to date. We found that M. bovis can be killed directly by the complement system
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