Characterization of the metabolism inhibition antigen of Mycoplasma arthritidis

Abstract

The Mycoplasma arthritidis antigen(s) responsible for eliciting metabolism-inhibiting antibodies in rabbits has been partially characterized. Metabolism-inhibiting activity was absorbed from rabbit antisera by intact M. arthrtidlis cells and membranes but much less so by the soluble cytoplasmic fraction, indicating that the antigen is located on the outer membrane surface. It was stable to periodate and lipid extraction but labile to heat and proteolytic enzymes, indicating that it is protein in nature. Finally, it is most likely a tightly bound integral rather than a peripheral membrane protein, since it was not extracted by low-ionic-strength solutions or by the nonionic detergents Triton X-100, Nonidet P-40, and Tween 20. It was solubilized by both the anionic agent sodium deoxycholate and the zwitterionic detergent Zwittergent14. Two monoclonal antibodies with metabolism-inhibiting activity were produced. One recognized a 45,000-dalton surface protein; however, the other recognized an antigen which is probably of cytoplasmic origin, indicating that more than one cell component may be involved in the metabolism-inhibiting antibody response.

Comparison of Mycoplasma arthritidis strains by enzyme-linked immunosorbent assay, immunoblotting, and DNA restriction analysis

“…These studies have uncovered cross-reactivities between M. arthritidis and Mycoplasma pulmonis (31) and between M. arthritidis and various rat tissues (41,45). In addition, antigens that serve as targets for metabolism-inhibiting antibodies (50) and two surface proteins involved in adherence to host tissues (52) were identified. Droesse et al (10)(11)(12) showed that several M. arthritidis surface proteins undergo rapid spontaneous size and phase variations both in vivo and in vitro, which could possibly complicate immune recognition.…”

Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis

“…Analysis of antigens by specific antibodies (and in some cases monoclonal antibodies [MAbs]) has been useful in defining potentially important surface structures of some mycoplasma species (10,14,26); this includes the initial characterization of M. hyorhinis surface components in our own laboratory (31,32) and by others (13). Elucidation of mycoplasma surface antigen structure is also important in light of the implied involvement of surface antigens in antibody-mediated metabolic and growth inhibition tests, which are widely used to classify mycoplasma species (11,24).…”

Mechanisms of attachment of Mycoplasma arthritidis to host cells in vitro

“…After overnight incubation, the culture was frozen at -70°C in 1.5to 2-ml aliquots. Surface-MYCOPL^ASMA4 ARTHRITIDIS ADHERENCE 2671 oriented membrane proteins were labeled with 125I (43) as described by Hubbard and Cohn (17). Labeled proteins were visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

“…Antisera. Rabbit hyperimmune antisera against M. arthintidis 158plOp9, 14124plO, PG6, and H606 were prepared as described previously (42,43). Monoclonal antibodies (MAbs) against M. arthritidis 158plOp9 were prepared from BALB/c mice and purified from mouse ascites fluids by protein A-Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) affinity chromatography by standard procedures.…”

“…Western and colony blotting. MAbs were tested at a dilution of 1:160 against total solubilized M. arthritidis proteins separated by SDS-PAGE on either 7.5 or 12.5% (wt/vol) gels and blotted onto nitrocellulose membranes (43). Bands were visualized with peroxidase-conjugated sheep anti-mouse IgG (heavyand light-chain specific; Organon Teknika), diluted 1:1,600, as the second antibody and a 4-chloro-1-naphthol-H202 substrate.…”